Antigen presenting system and methods for activation of T-cells

ABSTRACT

The present invention relates to synthetic antigen-presenting matrices, their methods of making and their methods of use. One such matrix is cells that have been transfected to produce MHC antigen-presenting molecules and assisting molecules such as co-stimulatory molecules. The matrices can be used to activate CD8 +  T-cells to produce cytokines and become cytotoxic.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a division of U.S. patent application Ser. No.08/913,612, filed on Sep. 8, 1997, now U.S. Pat. No. 6,461,867, which isa national stage application of PCT/US96/03249, filed Mar. 8, 1996,which is a continuation-in-part of U.S. patent application Ser. No.08/400,338 filed Mar. 8, 1995, now abandoned.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with the support of the Government of the UnitedStates of America under Contract No. CA 38355 by the National Institutesof Health, and the Government of the United States of America hascertain rights in the invention.

TECHNICAL FIELD

The present invention relates to materials and methods of activatingT-cells with specificity for particular antigenic peptides, the use ofactivated T-cells in vivo for the treatment of a variety of diseaseconditions, and compositions appropriate for these uses.

BACKGROUND

The efficiency with which the immune system cures or protectsindividuals from infectious disease has always been intriguing toscientists, as it has been believed that it might be possible toactivate the immune system to combat other types of diseases. Suchdiseases include cancer, AIDS, hepatitis and infectious disease inimmunosuppressed patients. While various procedures involving the use ofantibodies have been applied in those types of diseases, few if anysuccessful attempts using cytotoxic T-cells have been recorded.Theoretically, cytotoxic T-cells would be the preferable means oftreating the types of disease noted above. However, no procedures havebeen available to specifically activate cytotoxic T-cells.

Cytotoxic T-cells, or CD8⁺ cells (i.e., cells expressing the moleculeCD8) as they are presently known, represent the main line of defenseagainst viral infections. CD8⁺ lymphocytes specifically recognize andkill cells which are infected by a virus. Thus, the cost of eliminatinga viral infection is the accompanying loss of the infected cells. TheT-cell receptors on the surface of CD8⁺ cells cannot recognize foreignantigens directly. In contrast to antibodies, antigen must first bepresented to the receptors.

The presentation of antigen to CD8⁺ T-cells is accomplished by majorhistocompatibility complex (MHC) molecules of the Class I type. Themajor histocompatibility complex (MHC) refers to a large genetic locusencoding an extensive family of glycoproteins which play an importantrole in the immune response. The MHC genes, which are also referred toas the HLA (human leucocyte antigen) complex, are located on chromosome6 in humans. The molecules encoded by MHC genes are present on cellsurfaces and are largely responsible for recognition of tissuetransplants as “non-self”. Thus, membrane-bound MHC molecules areintimately involved in recognition of antigens by T-cells.

MHC products are grouped into three major classes, referred to as I, II,and III. T-cells that serve mainly as helper cells express CD4 andprimarily interact with Class II molecules, whereas CD8-expressingcells, which mostly represent cytotoxic effector cells, interact withClass I molecules.

Class I molecules are membrane glycoproteins with the ability to bindpeptides derived primarily from intracellular degradation of endogenousproteins. Complexes of MHC molecules with peptides derived from viral,bacterial and other foreign proteins comprise the ligand that triggersthe antigen responsiveness of T-cells. In contrast, complexes of MHCmolecules with peptides derived from normal cellular products play arole in “teaching” the T-cells to tolerate self-peptides, in the thymus.Class I molecules do not present entire, intact antigens; rather, theypresent peptide fragments thereof, “loaded” onto their “peptide bindinggroove”.

For many years, immunologists have hoped to raise specific cytotoxiccells targeting viruses, retroviruses and cancer cells. While targetingagainst viral diseases in general may be accomplished in vivo byvaccination with live or attenuated vaccines, no similar success hasbeen achieved with retroviruses or with cancer cells. Moreover, thevaccine approach has not had the desired efficacy in immunosuppressedpatients. At least one researcher has taken the rather non-specificapproach of “boosting” existing CD8⁺ cells by incubating them in vitrowith IL-2, a growth factor for T-cells. However, this protocol (known asLAK cell therapy) will only allow the expansion of those CD8⁺ cellswhich are already activated. As the immune system is always active forone reason or another, most of the IL-2 stimulated cells will beirrelevant for the purpose of combatting the disease. In fact, it hasnot been documented that this type of therapy activates any cells withthe desired specificity. Thus, the benefits of LAK cell therapy arecontroversial at best, and the side effects are typically so severe thatmany studies have been discontinued.

Several novel molecules which appear to be involved in the peptideloading process have recently been identified. It has also been notedthat Class I molecules without bound peptide (i.e., “empty” molecules)can be produced under certain restrictive circumstances. These “empty”molecules are often unable to reach the cell surface, however, as ClassI molecules without bound peptide are very thermolabile. Thus, the“empty” Class I molecules disassemble during their transport from theinterior of the cell to the cell surface.

The presentation of Class I MHC molecules bound to peptide alone hasgenerally ineffective in activating CD8⁺ cells. In nature, the CD8⁺cells are activated by antigen-presenting cells which present not only apeptide-bound Class I MHC molecule, but also a costimulatory molecule.Such costimulatory molecules include B7 which is now recognized to betwo subgroups designated as B7.1 and B7.2. It has also been found thatcell adhesion molecules such as integrins assist in this process.

When the CD8⁺ T-cell interacts with an antigen-presenting cell havingthe peptide bound by a Class I MHC and costimulatory molecule, the CD8⁺T-cell is activated to proliferate and becomes an armed effector T-cell.See, generally, Janeway and Travers, Immunobiology, published by CurrentBiology Limited, London (1994), incorporated by reference.

Accordingly, what is needed is a means to activate T-cells so that theyproliferate and become cytotoxic. It would be useful if the activationcould be done in vitro and the activated cytotoxic T-cells reintroducedinto the patient. It would also be desirable if the activation could bedone by a synthetic antigen-presenting matrix comprised of a materialsuch as cells which not only presents the selected peptide, but alsopresents other costimulatory factors which increase the effectiveness ofthe activation.

It would also be advantageous if it was possible to select the peptideso that substantially only those CD8⁺ cells cytotoxic to cellspresenting that peptide would be activated.

BRIEF SUMMARY OF THE INVENTION

The present invention relates to a synthetic antigen-presenting systemfor presenting an MHC molecule complexed to a peptide and an assistingmolecule to a T-cell to activate the T-cell.

In one embodiment, the system relates to a synthetic antigen-presentingmatrix having a support and at least the extracellular portion of aClass I MHC molecule capable of binding to a selected peptide operablylinked to the support. The matrix also includes an assisting moleculeoperably linked to the support. The assisting molecule acts on areceptor on the CD8⁺ T-cell. The MHC and assisting molecules are presentin sufficient numbers to activate a population of T-cell lymphocytesagainst the peptide when the peptide is bound to the extracellularportion of the MHC molecule.

It has been found that an antigen-presenting matrix having both an MHCmolecule or a portion of a MHC molecule together with an assistingmolecule, provides a synergistic reaction in activating T-celllymphocytes against the peptide. Examples of assisting molecules arecostimulatory molecules such as B7.1 and B7.2 or adhesion molecules suchas ICAM-1 and LFA-3. The extracellular portion of such costimulatorymolecules can also be used. Another type of costimulatory molecule isone that reacts with the CD28 molecule such as anti-CD28 antibodies orfunctional portions thereof, e.g. Fab portions.

It has been found that a specifically effective synergistic reactionresults from an antigen-presenting matrix having MHC molecules boundwith a peptide, a costimulatory molecule, and an adhesion molecule. Inparticular, a highly effective synergistic generation of cytotoxicT-cell activity results from the combination of 27.1 and ICAM-1.

The support used for the matrix can take several different forms.Examples for the support include solid support such as metals orplastics, porous materials such as resin or modified cellulose columns,microbeads, microtiter plates, red blood cells and liposomes.

Another type of support is a cell fragment, such as a cell membranefragment or an entire cell. In this embodiment, the matrix is actuallycells which have been transfected to present MHC molecules and assistingmolecules on the cell surface to create an antigen-presenting cell(APC). This is done by producing a cell line containing at least oneexpressible Class I MHC nucleotide sequence for the MHC heavy chain,preferably a cDNA sequence, operably linked to a first promoter and anexpressible β2 microglobulin nucleotide sequence operably linked to asecond promoter. The MHC heavy chain and the β2 microglobulin associatetogether form the MHC molecule which binds to the peptide. The MHCprotein binds with the antigenic peptide and presents it on the surfaceof the cell. The cell also includes a gene for a nucleotide sequence ofan assisting molecule operably linked to a third promoter. The assistingmolecule is also presented on the surface of the cell. These moleculesare presented on the surface of the cell in sufficient numbers toactivate a population of T-cell lymphocytes against the peptide when thepeptide is bound to the complexes. Other molecules on the surface of acell or cell fragment such as carbohydrate moieties may also providesome costimulation to the T-cells.

The cell line is synthetic in that at least one of the genes describedabove is not naturally present in the cells from which the cell line isderived. It is preferable to use a poikilotherm cell line because MHCmolecules are thermolabile. A range of species are useful for thispurpose. See, for example, U.S. Pat. No. 5,314,813 to Petersen et al.which discusses numerous species for this use and is incorporated byreference. It is preferred to use eukaryotic cells and insect cells inparticular.

In one embodiment, it is particularly preferred to have at least twoassisting molecules, one being a costimulatory molecule and the otherbeing an adhesion molecule. It has been found that this combination hasa synergistic effect, giving even greater T-cell activation than eitherof the individual molecules combined. It has also been found to beadvantageous and preferable to have at least one of the transfectedgenes under control of an inducible promoter.

Using the present invention, it is possible to introduce the peptide tothe cell while it is producing MHC molecules and allow the peptide tobind the MHC molecules while they are still within the cell.Alternatively, the MHC molecules can be expressed as empty molecules onthe cell surface and the peptide introduced to the cells after themolecules are expressed on the cell surface. In this latter procedure,the use of poikilotherm cells is particularly advantageous because emptyMHC molecules, those not yet complexed or bound with peptides, arethermolabile.

Class I MHC molecules have been expressed in insect cells such asDrosophila melanogaster (fruit fly) cells. Since Drosophila does nothave all the components of a mammalian immune system, the variousproteins involved in the peptide loading machinery should be absent fromsuch cells. The lack of peptide loading machinery allows the Class Imolecules to be expressed as empty molecules at the cell surface.

Another advantage of using insect cells such as the Drosophila system isthat Drosophila cells prefer a temperature of 28° C. rather than 37° C.This fact is very important, because empty Class I molecules arethermolabile and tend to disintegrate at 37° C. By incubating the ClassI-expressing Drosophila cells with peptides that can bind to the Class Imolecule, it is possible to get virtually every Class I molecule tocontain one and the same peptide. The cells are accordingly verydifferent from mammalian cells, where the Class I molecules contain manydifferent types of peptides, most of which are derived from our own,innocuous cellular proteins.

The present invention also relates to methods for producing activatedCD8⁺ cells in vitro. One method comprises contacting, in vitro, CD8⁺cells with one of the antigen-presenting matrices described above for atime period sufficient to activate, in an antigen-specific manner, theCD8⁺ cells. The method may further comprise (1) separating the activatedCD8⁺ cells from the antigen-presenting matrix; (2) suspending theactivated CD8⁺ cells in an acceptable carrier or excipient; and (3)administering the suspension to an individual in need of treatment. Theantigens may comprise native or undegraded proteins or polypeptides, orthey may comprise antigenic polypeptides which have been cleaved intopeptide fragments comprising at least 8 amino acid residues prior toincubation with the human Class I MHC molecules.

In another variation, the invention relates to methods treatingconditions in patients and specifically killing target cells in a humanpatient. The method comprises (1) obtaining a fluid sample containingresting or naive CD8⁺ cells from the patient; (2) contacting, in vitro,the CD8⁺ cells with an antigen-presenting matrix for a time periodsufficient to activate, in an antigen-specific manner, the CD8⁺ cells;and (3) administering the activated CD8⁺ cells to the patient. Forexample, the use of tumor specific peptides allows for the treatment oftumor related diseases by producing cytotoxic activated CD8⁺ T-cells.The invention also relates to the method of treating a medical conditionby administration of an antigen-presenting matrix in a suitablesuspension. In various embodiments the condition may comprise cancer,tumors, neoplasia, viral or retroviral infection, autoimmune orautoimmune-type conditions. In one embodiment, the method ofadministering the matrix comprises intravenous injection.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-3 diagram the construction of expression plasmids pRmHa-2 andpRmHa-3. In FIG. 1, pRmHa-2 construction is shown; in FIG. 2, pRmHa-3construction is shown; and in FIG. 3, the pRmHa-3 vector is illustrated,showing the restriction, polylinker, promoter, and polyadenylationsites, as well as a site at which a nucleotide sequence may be insertedfor expression;

FIGS. 4 and 5 show peptide-induced thermostabilization of HLA B27 andHLA A2.1 expressed on the surface of Drosophila cells by HIV peptides.The mean fluorescence of each cell population is shown plotted againstthe incubation conditions;

FIG. 6 illustrates data from an experiment designed to determine whetherinsect cells can process antigen and load it onto the Class I molecules,and whether the latter can present either endogenously or exogenouslyderived antigen to T-cells. Schneider 2 (SC2) or 3T3 cells transfectedwith K^(b)/β2 were incubated with ovalbumin protein (OvPro) or ovalbuminpeptide, OVA 24 (OvPep) in isotonic (Iso) or hypertonic (Hyp) media.(Murine cell line BALB/3T3 is available from the ATCC under accessionnumber CCL 163.) After treatment, cells were cocultured with the T-cellhybridoma B3/CD8. B3/CD8 is a T-cell hybridoma between B3 (Carbone, etal., J. Exp. Med. 169: 603-12 (1989)), cytotoxic T-cell specific forovalbumin peptide 253-276 presented by H-2K^(b) Class I molecules, andCD8-bearing IL-2-secreting cell line. Upon antigenic stimulation, B3/CD8produces IL-2, measured by ³H hymidine incorporation in IL-2-dependentcell line CTLL (Gillis, et al., J. Immunol. 120: 2027 91978)). Thus, bymeasuring the amount of IL-2 produced, one can assay for T-cellrecognition. The supernatant from the cocultures were analyzed for IL-2by ³H thymidine incorporation by the IL-2-dependent cell line CTLL (ATCCNo. TIB 214). The amount of ³H thymidine incorporated is plotted againstthe initial cell treatments;

FIG. 7 illustrates the expression of B7.1, ICAM-1 and MHC on the surfaceof transfected Drosophila (fly) cells according to the presentinvention;

FIG. 8 is a graph showing results of a fluorescence-activated cellsorter experiment using recombinant L^(d) mouse MHC linked to red bloodcells;

FIG. 9 is a graph showing results of a fluorescence-activated cellsorter experiment using recombinant K^(b) mouse MHC linked to red bloodcells;

FIG. 10 is a graph demonstrates binding of recombinant K^(b) tomicrotiter plates by use of labeled antibodies;

FIG. 11 is a series of graphs showing the results fromfluorescence-activated cell sorter experiments demonstrating theexpression of CD69 and CD25 on CD8⁺ 2C cells stimulated with transfectedDrosophila cells;

FIGS. 12A-B is a pair of bar graphs showing IL-2-dependent proliferativeresponses of CD8⁺ 2C cells elicited by peptides presented by Drosophilacells transfected with L^(d) only;

FIG. 13 is a graph showing the influence of peptide concentration on day3 proliferative responses of CD8⁺ 2C cells elicited by peptidespresented by transfected Drosophila cells;

FIGS. 14A-B is a pair of graphs showing the influence ofantigen-presenting cells dose on day 3 proliferative response and IL-2production of CD8⁺ 2C cells elicited by peptides presented bytransfected Drosophila cells;

FIGS. 15A-C is a series of graphs showing the influence of peptideconcentration on the proliferative response of CD8⁺ 2C cells elicited byDrosophila cells transfected with L^(d).B7, L^(d).ICAM andL^(d).B7.1CAM;

FIGS. 16A-D is a series of graphs showing the kinetics of theproliferative response of CD8⁺ and CD8.sup.-2C cells elicited byDrosophila cells transfected with L^(d), L^(d).B7, L^(d).ICAM andLd.B7.1CAM plus QL9 peptide;

FIGS. 17A-C is a series of graphs showing CTL activity of CD8⁺ 2C cellsstimulated by Drosophila cells transfected with L^(d).B7, L^(d).B7.1CAMor L^(d).ICAM antigen-presenting cells plus QL9 peptide (10 μM) in theabsence of exogenous cytokines;

FIGS. 18A-B is a pair of graphs showing CTL activity of CD8⁺ 2C cellsstimulated by Drosophila cells transfected with L^(d).ICAMantigen-presenting cells plus QL9 peptide (10 μM) in the absence (left)or presence (right) of exogenous IL-2 (20 u/ml);

FIGS. 19A-B is a pair of graphs showing the proliferative response ofnormal (non-transgenic) CD8⁺ T-cells to peptides presented bytransfected Drosophila cells (left panel) and the response elicited bygraded doses of N B6 and 2C B6 CD8⁺ cells cultured with 5×10⁹ B10.D2(L^(d)) spleen cells (2000 cGy) in the absence of peptides for 3 dayswithout the addition of exogenous cytokines (right panel);

FIG. 20 is a graph showing stimulated mitogenesis of purified 2C+T-cells cultured (50,000 cells per well) in plates coated withimmobilized molecules with peptide QL9 (solid line=L^(d) and anti-CD28antibody, broken line=L^(d) only);

FIG. 21 is a bar graph showing stimulated mitogenesis of purified 2C+T-cells at day 5 in culture, with various indicated peptides, culturedin 96-well plates coated with L^(d) and anti-CD28 antibody (hatchedbars=no IL-2, black bars=IL-2 added);

FIGS. 22A-B is a graphical representation of the results ofcytofluorometric analysis of cells recovered after 12 days of culture inplates coated with L^(d) and anti-CD28 antibody and exposed to peptideQL9, stained using the 2C T-cell receptor specific antibody 1B2(M2=positive staining cells, M1=negative staining cells);

FIGS. 23A-B is a graphical representation of the results ofcytofluorometric analysis of cells recovered after 12 days of culture inplates coated with L^(d) and anti-CD28 antibody and exposed to peptidep2Ca, stained using the 2C T-cell receptor specific antibody 1B2(M2=positive staining cells, M1=negative staining cells);

FIGS. 24A-B is a graphical representation of the results ofcytofluorometric analysis of cells recovered after 12 days of culture inplates coated with L^(d) and anti-CD28 antibody and exposed to peptideSL9, stained using the 2C T-cell receptor specific antibody 1B2(M2=positive staining cells, M1=negative staining cells);

FIG. 25 is a graph showing cytolysis of target cells by activatedT-cells (solid line=peptide QL9, broken line=control peptide LCMV);

FIGS. 26A-C is a graphical representation of the cytotoxic lysisresulting from activation of human CD8⁺ T-cells with antigen-presentingcells loaded with influenza matrix peptide;

FIGS. 27A-C is a graphical representation of the cytotoxic lysisresulting from activation of human CD8⁺ T-cells with antigen-presentingcells loaded with HIV-RT peptide; and

FIGS. 28A-C is a graphical representation of the cytotoxic lysisresulting from activation of human CD8⁺ T-cells with antigen-presentingcells loaded with tyrosinase peptide.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a synthetic antigen-presenting systemwhich can be used to activate T-cell lymphocytes. The activated CD8⁺T-cells proliferate, produce cytokines, become cytotoxic or somecombination of these results. In one preferred embodiment, the systemactivates cytotoxic CD8⁺ cells which then proliferate and then areactivated to seek out and destroy target cells. The present inventioncan be used to activate T-cells in vitro and the activated T-cells arethen returned to the patient from which they were originally derived ormay be used in vivo activation of T-cells.

The synthetic antigen-presenting system of the present invention has twomajor components. The first component is at least the extracellularportion of the Class I MHC molecule which is capable of binding to aselected peptide. The second major component is an assisting moleculewhich assists in the activation of T-cells. In each case, anextracellular portion of a larger molecule can used, but in certainembodiments, the entire molecules are used.

For ease of description, MHC molecules will be discussed generally, withthe understanding that an extracellular portion of the MHC molecule maybe used. The portion of the MHC molecule necessary for the presentinvention is the part which binds to the selected peptide and presentsthe peptide to the T-cell.

The peptide is selected to activate the appropriate T-cell, depending onthe treatment to be conducted. For example, in the treatment ofparticular cancers, certain antigenic peptides are presented on thesurface of the cancer cells which will react with activated T-cells.Thus, it is appropriate to use a peptide selected to activate theappropriate T-cells that will then bind with and destroy the cancercells.

The present invention allows the MHC molecules to be produced by cellswith the peptide already complexed with the MHC molecule or to produceempty MHC molecules which do not yet have a peptide complexed with them.This latter embodiment is particularly useful since it allows thepeptide to be chosen after the MHC molecules are prepared.

A Class I MHC molecule includes a heavy chain, sometimes referred to asan alpha chain, and a β-2 microglobulin. As discussed herein, theextracellular portion of the Class I MHC molecule is made up of anextracellular portion of an MHC heavy chain together with the β-2microglobulin.

In preparing the extracellular portions of MHC to be linked to asupport, soluble molecules are prepared as discussed below. Thesemolecules generally lack the transmembrane and cytoplasmic domain in theMHC molecule.

The assisting molecule helps facilitate the activation of the T-cellwhen it is presented with a peptide/MHC molecule complex. The presentinvention includes two major categories of assisting molecules. Thefirst category is composed of costimulatory molecules such as B7.1(previously known as B7 and also known as CD80) and B7.2 (also known asCD86) which binds to CD28 on T-cells. Other costimulatory molecules areanti-CD28 antibodies or the functional portions of such antibodies, e.g.Fab portions that bind to CD28.

The other major category of assisting molecules of the present inventionare adhesion molecules. These include the various ICAM molecules, whichinclude ICAM-1, ICAM-2, ICAM-3 and LFA-3. It has been found that thecombination of a peptide bound to an MHC molecule used in conjunctionwith one of these assisting molecules activates the T-cells to an extentpreviously not seen.

An even greater synergistic reaction has been achieved by using apeptide-bound MHC molecule in conjunction with both a costimulatorymolecule and an adhesion molecule. This has been found to beparticularly effective in producing cytotoxic CD8⁺ cells.

In accordance with the present invention, the MHC molecule and theassisting molecule are operably linked to a support such that the MHCand assisting molecules are present in sufficient numbers to activate apopulation of T-cells lymphocytes against the peptide when the peptideis bound to the extracellular portion of the MHC molecule. The peptidecan be bound to the MHC molecule before or after the MHC molecule islinked to the support.

The support can take on many different forms. It can be a solid supportsuch as a plastic or metal material, it can be a porous material such ascommonly used in separation columns, it can be a liposome or red bloodcell, or it can even be a cell or cell fragment. As discussed in moredetail below, in the case where a cell serves as a support, the MHC andassisting molecules can be produced by the cell. The MHC molecule isthen linked to the cell by at least the transmembrane domain if not alsothe cytoplasmic domain which would not be present in a soluble form ofMHC.

The extracellular portions of MHC molecule and assisting molecule can belinked to a support by providing an epitope which reacts to an antibodyimmobilized on the support. In addition, the MHC or assisting moleculescan be produced with or linked to (His)₆ which would react with nickelin forming part of the support. Other means to immobilize or link MHCmolecules to a support are well known in the art.

As discussed above, the support can be a cell membrane or an entirecell. In such a case, an eukaryotic cell line is modified to become asynthetic antigen-presenting cell line for use with T-cell lymphocytes.For ease of description, antigen-presenting cells (APC) will also becalled stimulator cells. Because empty MHC molecules are thermolabile,it is preferred that the cell culture be poikilotherm and various celllines are discussed in detail below.

A culture of cells is first established. The culture is then transfectedwith an expressible Class I MHC heavy chain gene which is operablylinked to a promoter. The gene is chosen so that it is capable ofexpressing the Class I MHC heavy chain. The cell line is alsotransfected with an expressible β-2 microglobulin gene which is operablylinked to a second promoter. The gene is chosen such it is capable ofexpressing β-2 microglobulin that forms MHC molecules with the MHC heavychain. In the case of soluble extracellular portions of MHC molecules tobe used with solid supports and the like, a truncated MHC heavy chaingene is used as discussed in more detail below.

The culture is also transfected with an expressible assisting moleculegene operably linked to a third promoter. The assisting molecule gene iscapable of being expressed as an assisting molecule which interacts withthe molecule on the T-cell lymphocytes. As discussed below, each ofthese genes can be transfected using various methods, but the preferredmethod is to use more than one plasmid.

The cell line transfected is chosen because it lacks at least one of thegenes being introduced. It has been found that insect cells areadvantageous not only because they are poikilothermic, but because theylack these genes and the mechanisms which would otherwise produce MHCmolecules bound to peptides. This allows for greater control over theproduction of peptide-bound MHC molecules, and the production of emptyMHC molecules. The MHC heavy chain is preferably from a differentspecies, more preferably, a homeotherm such as mammals and, optimally,humans.

The preferred cell line is a stable poikilotherm cell line that has thefirst promoter being inducible to control the expression of the MHCheavy chain. It is preferred that the cell assembles empty MHC moleculesand presents them on the cell surface so that the peptides can be chosenas desired.

The resulting MHC molecules bind to the peptide and are present insufficient numbers with the assisting molecules on the surface of thecell to activate a population of T-cell lymphocytes against the peptidewhen the peptide is bound to the MHC cells.

In a further embodiment, a second assisting molecule gene is alsotransfected into the cell culture. In this case, the first assistingmolecule gene can be for a costimulatory molecule and the secondassisting molecule gene can be for an adhesion molecule.

It is preferred that at least one of the genes and, in particular, theMHC heavy chain gene be linked to an inducible promoter. This allowscontrol over the production of MHC molecules so that they are onlyproduced at a time when the peptide of interest is available andpresented in the culture to react with the produced MHC molecules. Thisminimizes undesirable MHC molecule/peptide complexes.

Where the cell line already produces one or more of the desiredmolecules, it is only necessary to transfect the culture with anexpressible gene for the gene which is lacking in the cells. Forexample, if the cells already present the MHC molecules on theirsurface, it is only necessary to transfect the culture with anexpressible gene for the assisting molecule.

The peptide can be introduced into the cell culture at the time thecells are producing MHC molecules. Through methods such as osmoticshock, the peptides can be introduced in the cell and bind to theproduced MHC molecules. Alternatively, particularly in the casepoikilotherm cell lines, the MHC molecules will be presented empty onthe cell surface. The peptide can then be added to the culture and boundto the MHC molecules as desired.

After the cells are produced having MHC and assisting molecules on theirsurfaces, they can be lyophilized and the fragments of the cells used toactivate the population of T-cell lymphocytes.

Transfected cultures of cells can be used to produced extracellularportions of MHC molecules and assisting molecules. The use ofextracellular portions n conjunction with supports such as solidsupports has certain advantages of production. Where living cells areused to provide a synthetic antigen-presenting cell, at least threegenes, two to produce the MHC molecule and one for the assistingmolecule must be introduced to the cell. Often, additional genes such asfor antibiotic resistance are also transfected.

Where a solid support system is being used, one cell line can producethe extracellular portions of MHC molecules while another cell lineproduces the extracellular portion of the assisting molecule. The MHCmolecule portions and the assisting molecule portions can then beharvested from their respective cultures. The molecules are then linkedto an appropriate support in sufficient numbers to activate a populationof T-cell lymphocytes against a peptide when the peptide is bound to theextracellular portion of the MHC molecule. From a production standpoint,two different cultures can be used, but it is also possible to use thesame culture, however, requiring that the culture be transfected withthe additional gene for the extracellular portion of the assistingmolecule.

A further modification of this embodiment is to provide a third cultureof cells which is transfected with an expressible second assistingmolecule gene. In this example, the second culture of cells producesextracellular portions of the costimulatory molecule while the thirdculture of cells produce an extracellular portion of an adhesionmolecule. The adhesion molecule portions are harvested and linked to thesupport.

The present invention also relates to a method for activating CD8⁺T-cells against a selected peptide. The method relates to providing acell line presenting MHC molecules binding a peptide and assistingmolecules on their surfaces. Naive CD8⁺ T-cells can be obtained byremoval from a patient to be treated. The cultured cells are thencontacted with the CD8⁺ T-cells for a sufficient period of time toactivate the CD8⁺ T-cell lymphocytes resulting in proliferation andtransforming the T-cells into armed effector cells.

The activated CD8⁺ T-cells can then be separated from the cell line andput into a suspension in an acceptable carrier and administered to thepatient. An alternative method involves the use of the syntheticantigen-presenting matrix to activate the CD8⁺ cells.

It is preferred that human genes are used and, therefore, human moleculeanalogs are produced. As shown in prior U.S. Pat. No. 5,314,813, murinesystems provide particularly useful models for testing the operation ofT-cell activation and demonstrate the applicability of the process forhuman systems. See also Sykulev et al., Immunity 1: 15-22 (1994).

Human Class I MHC Molecules

Class I MHC molecules comprise a heavy chain and a β-microglobulinprotein. A human Class I MHC heavy chain of the present invention isselected from the group comprising HLA-A, HLA-B, HLA-C, HLA-E, HLA-F,and HLA-G, and more preferably, from the group comprising HLA-A, HLA-B,and HLA-C. The heavy chains are useful in either soluble or insolubleform. In the soluble (“sol”) form, a stop codon is engineered into thenucleotide sequence encoding the HLA molecule of choice preceding thetransmembrane domain.

While it is possible to isolate nucleotide sequences encoding humanClass I MHC heavy chains from known, established cell lines carrying theappropriate variants—e.g., transformed cell lines JY, BM92, WIN, MOC,and MG—it is more practical to synthesize the nucleotide sequence from aportion of the gene via polymerase chain reaction (PCR), using theappropriate primers. This method has been successfully used to clonefull-length HLA cDNA; for example, the sequences for HLA-A25, HLA-A2,HLA-B7, HLA-B57, HLA-B51, and HLA-B37 are deposited in the GenBankdatabase under accession nos. M32321, M32322, M32317, M32318, M32319 andM32320, respectively. Known, partial and putative HLA amino acid andnucleotide sequences, including the consensus sequence, are published(see, e.g., Zemmour and Parham, Immunogenetics 33: 310-320 (1991)), andcell lines expressing HLA variants are known and generally available aswell, many from the American Type Culture Collection (“ATCC”).Therefore, using PCR, it is possible to synthesize human Class IMHC-encoding nucleotide sequences which may then be operatively linkedto a vector and used to transform an appropriate cell and expressedtherein.

Particularly preferred methods for producing the Class I MHC heavychain, β-2 microglobulin proteins and assisting molecules of the presentinvention rely on the use of preselected oligonucleotides as primers ina polymerase chain reaction (PCR) to form PCR reaction products asdescribed herein. Gene preparation is typically accomplished by primerextension, preferably by primer extension in a polymerase chain reaction(PCR) format.

If the genes are to be produced by (PCR) amplification, two primers,i.e., a PCR primer pair, must be used for each coding strand of nucleicacid to be amplified. (For the sake of simplicity, synthesis of anexemplary HLA heavy chain variant sequence will be discussed, but it isexpressly to be understood that the PCR amplification method describedis equally applicable to the synthesis of β-2 microglobulin,costimulatory molecules, adhesion molecules, and all HLA variants,including those whose complete sequences are presently unknown.)

The first primer becomes part of the antisense (minus or complementary)strand and hybridizes to a nucleotide sequence conserved among HLA (plusor coding) strands. To produce coding DNA homologs, first primers aretherefore chosen to hybridize to (i.e. be complementary to) conservedregions within the MHC genes, preferably, the consensus sequence orsimilar, conserved regions within each HLA group—i.e., consensussequences within HLA-A, HLA-B, HLA-C, and the less-polymorphic groups,HLA-E, -F, and -G.

Second primers become part of the coding (plus) strand and hybridize toa nucleotide sequence conserved among minus strands. To produce theHLA-coding DNA homologs, second primers are therefore chosen tohybridize with a conserved nucleotide sequence at the 5′ end of theHLA-coding gene such as in that area coding for the leader or firstframework region. In the amplification of the coding DNA homologs theconserved 5′ nucleotide sequence of the second primer can becomplementary to a sequence exogenously added using terminaldeoxynucleotidyl transferase as described by Loh et al., Science 243:217-220 (1989). One or both of the first and second primers can containa nucleotide sequence defining an endonuclease recognition site. Thesite can be heterologous to the immunoglobulin gene being amplified andtypically appears at or near the 5′ end of the primer.

The high turn over rate of the RNA polymerase amplifies the startingpolynucleotide as has been described by Chamberlin et al., The Enzymes,ed. P. Boyer, PP. 87-108, Academic Press, New York (1982). Anotheradvantage of T7 RNA polymerase is that mutations can be introduced intothe polynucleotide synthesis by replacing a portion of cDNA with one ormore mutagenic oligodeoxynucleotides (polynucleotides) and transcribingthe partially-mismatched template directly as has been previouslydescribed by Joyce et al., Nuc. Acid Res. 17: 711-722 (1989).Amplification systems based on transcription have been described byGingeras et al., in PCR Protocols, A Guide to Methods and Applications,pp 245-252, Academic Press, Inc., San Diego, Calif. (1990).

PCR amplification methods are described in detail in U.S. Pat. Nos.4,683,192, 4,683,202, 4,800,159, and 4,965,188, and at least in severaltexts including “PCR Technology: Principles and Applications for DNAAmplification”, H. Erlich, ed., Stockton Press, New York (1989); and“PCR Protocols: A Guide to Methods and Applications”, Innis et al.,eds., Academic Press, San Diego, Calif. (1990). Various preferredmethods and primers used herein are described hereinafter and are alsodescribed in Nilsson, et al., Cell 58: 707 (1989), Ennis, et al., PNASUSA 87: 2833-7 (1990), and Zemmour, et al., Immunogenetics 33: 310-20(1991), for example. In particular, it is preferred to design primersfrom comparison of 5′ and 3′ untranslated regions of HLA alleles (e.g.,-A, -B, -C, -E, -F, or -G alleles), with selection of conservedsequences. Restriction sites may also be incorporated into the 5′ and 3′primers to enable the amplification products to be subcloned intosequencing or expression vectors. It may also be helpful to place a4-base spacer sequence proximal to the restriction site to improve theefficiency of cutting amplification products with enzymes.

The following primers are preferred for amplification of HLA-A, -B, -C,-E, -F, and -G cDNA, preferably in separate reactions. Resulting cDNAsmay then be cloned and sequenced as described herein. These primers areappropriate for use in amplifying all known and presently unknown typesof HLA.

HLA A 5′ primer: 5′ CC ACC ATG GCC GTC ATG GCG CCC 3′ (SEQ ID NO 1)3′ primer: 5′ GG TCA CAC TTT ACA AGC TCT GAG 3′ (SEQ ID NO 2) HLA B5′ primer: 5′ CC ACC ATG CTG GTC ATG GCG CCC 3′ (SEQ ID NO 3) 3′ primer:5′ GG ACT CGA TGT GAG AGA CAC ATC 3′ (SEQ ID NO 4) HLA C 5′ primer:5′ CC ACC ATG CGG GTC ATG GCG CCC 3′ (SEQ ID NO 5) 3′ primer: 5′ GG TCAGGC TTT ACA AGC GAT GAG 3′ (SEQ ID NO 6) HLA E 5′ primer: 5′ CC ACC ATGCGG GTA GAT GCC CTC C 3′ (SEQ ID NO 7) 3′ primer: 5′ GG TTA CAA GCT GTGAGA CTC AGA 3′ (SEQ ID NO 8) HLA F 5′ primer: 5′ CC ACC ATG GCG CCC CGAAGC CTC 3′ (SEQ ID NO 9) 3′ primer: 5′ GG TCA CAC TTT ATT AGC TGT GAG A3′ (SEQ ID NO 10) HLA G 5′ primer: 5′ CC ACC ATG GCG CCC CGA ACC CTC 3′(SEQ ID NO 11) 3′ primer: 5′ GG TCA CAA TTT ACA AGC CGA GAG 3′ (SEQ IDNO 12)

In preferred embodiments only one pair of first and second primers isused per amplification reaction. The amplification reaction productsobtained from a plurality of different amplifications, each using aplurality of different primer pairs, are then combined. However, thepresent invention also relates to DNA homolog production viaco-amplification (using two pairs of primers), and multiplexamplification (using up to about 8, 9 or 10 primer pairs).

In preferred embodiments, the PCR process is used not only to produce avariety of human Class I-encoding DNA molecules, but also to inducemutations which may emulate those observed in the highly-polymorphic HLAloci, or to create diversity from a single parental clone and therebyprovide a Class I MHC molecule-encoding DNA “library” having a greaterheterogeneity. In addition to the mutation inducing variations describedin the above referenced U.S. Pat. No. 4,683,195 and such as discussed inU.S. Pat. No. 5,314,813.

DNA Expression Vectors

A vector of the present invention is a nucleic acid (preferably DNA)molecule capable of autonomous replication in a cell and to which a DNAsegment, e.g., gene or polynucleotide, can be operatively linked so asto bring about replication of the attached segment. One of thenucleotide segments to be operatively linked to vector sequences encodesat least a portion of a mammalian Class I MHC heavy chain. Preferably,the entire peptide-coding sequence of the MHC heavy chain is insertedinto the vector and expressed; however, it is also feasible to constructa vector which also includes some non-coding MHC sequences as well.Preferably, non-coding sequences of MHC are excluded. Alternatively, anucleotide sequence for a soluble (“sol”) form of an Class I MHC heavychain may be utilized; the “sol” form differs from the non-sol form inthat it contains a “stop” codon inserted at the end of the alpha 3domain or prior to the transmembrane domain. Another preferred vectorincludes a nucleotide sequence encoding at least a portion of amammalian β-2 microglobulin molecule operatively linked to the vectorfor expression. Still another preferred vector includes a nucleotidesequence encoding at least a portion of a mammalian assisting moleculeoperably linked to the vector for expression. It is also feasible toconstruct a vector including nucleotide sequences encoding a Class I MHCheavy chain and a β-2 microglobulin and an assisting molecule, or somecombination of these.

A preferred vector comprises a cassette that includes one or moretranslatable DNA sequences operatively linked for expression via asequence of nucleotides adapted for directional ligation. The cassettepreferably includes DNA expression control sequences for expressing thepolypeptide or protein that is produced when a translatable DNA sequenceis directionally inserted into the cassette via the sequence ofnucleotides adapted for directional ligation. The cassette alsopreferably includes a promoter sequence upstream from the translatableDNA sequence, and a polyadenylation sequence downstream from themammalian MHC heavy chain sequence. The cassette may also include aselection marker, albeit it is preferred that such a marker be encodedin a nucleotide sequence operatively linked to another expression vectorsequence.

The choice of vector to which a cassette of this invention isoperatively linked depends directly, as is well known in the art, on thefunctional properties desired, e.g., vector replication and proteinexpression, and the host cell to be transformed, these being limitationsinherent in the art of constructing recombinant DNA molecules.

In various embodiments, a vector is utilized for the production ofpolypeptides useful in the present invention, including MHC variants andantigenic peptides. Exemplary vectors include the plasmids pUC8, pUC9,pUC18, pBR322, and pBR329 available from BioRad Laboratories (Richmond,Calif.), pPL and pKK223 available from Pharmacia (Piscataway, N.J.), andpBS and M13 mp19 (Stratagene, La Jolla, Calif.). Other exemplary vectorsinclude PCMU (Nilsson, et al., Cell 58: 707 (1989)). Other appropriatevectors may also be synthesized, according to known methods; forexample, vectors pCMU/K^(b) and pCMUII used in various applicationsherein are modifications of PCMUIV (Nilsson, et al., supra).

In addition, there is preferably a sequence upstream of the translatablenucleotide sequence encoding a promoter sequence. Preferably, thepromoter is conditional (e.g., inducible). A preferred conditionalpromoter used herein is a metallothionein promoter or a heat shockpromoter.

Vectors may be constructed utilizing any of the well-known vectorconstruction techniques. Those techniques, however, are modified to theextent that the translatable nucleotide sequence to be inserted into thegenome of the host cell is flanked “upstream” of the sequence by anappropriate promoter and, in some variations of the present invention,the translatable nucleotide sequence is flanked “downstream” by apolyadenylation site. This is particularly preferred when the “host”cell is an insect cell and the nucleotide sequence is transmitted viatransfection. Transfection may be accomplished via numerous methods,including the calcium phosphate method, the DEAE-dextran method, thestable transfer method, electroporation, or via the liposome mediationmethod. Numerous texts are available which set forth known transfectionmethods and other procedures for introducing nucleotides into cells;see, e.g., Current Protocols in Molecular Biology, John Wiley & Sons, NY(1991).

The vector itself may be of any suitable type, such as a viral vector(RNA or DNA), naked straight-chain or circular DNA, or a vesicle orenvelope containing the nucleic acid material and any polypeptides thatare to be inserted into the cell. With respect to vesicles, techniquesfor construction of lipid vesicles, such as liposomes, are well known.Such liposomes may be targeted to particular cells using otherconventional techniques, such as providing an antibody or other specificbinding molecule on the exterior of the liposome. See, e.g., A. Huang,et al., J. Biol. Chem. 255: 8015-8018 (1980). See, e.g., Kaufman, Meth.Enzymol. 185: 487-511 (1990).

In a preferred embodiment, the vector also contains a selectable marker.After expression, the product of the translatable nucleotide sequencemay then be purified using antibodies against that sequence. One exampleof a selectable marker is neomycin resistance. A plasmid encodingneomycin resistance, such as phshsneo, phsneo, or pcopneo, may beincluded in each transfection such that a population of cells thatexpress the gene(s) of choice may be ascertained by growing thetransfectants in selection medium.

A preferred vector for use according to the present invention is aplasmid; more preferably, it is a high-copy-number plasmid. It is alsodesirable that the vector contain an inducible promoter sequence, asinducible promoters tend to limit selection pressure against cells intowhich such vectors (which are often constructed to carry non-native orchimeric nucleotide sequences) have been introduced. It is alsopreferable that the vector of choice be best suited for expression inthe chosen host. If the host cell population is a Drosophila cellculture, then a compatible vector includes vectors functionallyequivalent to those such as p25-lacZ (see Bello and Couble, Nature 346:480 (1990)) or pRmHa-1, -2, or -3 (see Bunch, et al., Nucl. Acids Res.16: 1043-1061 (1988)). In the preferred embodiment, the vector ispRmHa-3, which is shown in FIG. 3. This vector includes ametallothionein promoter, which is preferably upstream of the site atwhich the MHC sequence is inserted, and the polyadenylation site ispreferably downstream of said MHC sequence. Insect cells and, inparticular, Drosophila cells are preferred hosts according to thepresent invention. Drosophila cells such as Schneider 2 (S2) cells havethe necessary trans-acting factors required for the activation of thepromoter and are thus even more preferred.

The expression vector pRmHa-3 is based on the bacterial plasmid pRmHa-1(FIG. 2), the latter of which is based on plasmid pUC18 and is depositedwith the American Type Culture Collection (ATCC, Rockville, Md.), havingthe accession number 37253. The pRmHa-3 vector contains the promoter,the 5′ untranslated leader sequence of the metallothionein gene(sequences 1-421, SEQ ID NO 13) with the R1 and Stu sites removed; seeFIG. 3). It also contains the 3′ portion of the Drosophila ADH gene(sequence #6435-7270, SEQ ID NO 14) including the polyadenylation site.Therefore, cloned DNA will be transcriptionally regulated by themetallothionein promoter and polyadenylated. Construction of the pRmHa-1plasmid is described in Bunch, et al., Nucl. Acids Res. 16: 1043-1061(1988). Construction of the pRmHa-3 and pRmHa-2 plasmids (the latter ofwhich has a metallothionein promoter sequence that may be removed as anEco RI fragment) is illustrated in FIGS. 1, 2, and 3. With regard topRmHa-3, a preferred plasmid for use according to the present invention,Pst I, Sph I and Hind III are in the promoter fragment and therefore arenot unique. Xba is in the ADH fragment (4 bases from its 3′ end) and isalso not unique. The following restriction sites are, however, unique inpRmHa-3: Eco RI, Sac I, Kpn I, Sma I, Bam HI, Sal I, Hinc 2, and Acc I.

A cassette in a DNA expression vector of this invention is the region ofthe vector that forms, upon insertion of a translatable DNA sequence, asequence of nucleotides capable of expressing, in an appropriate host, afusion protein of this invention. The expression-competent sequence ofnucleotides is referred to as a cistron. Thus, the cassette preferablycomprises DNA expression control elements operatively linked to one ormore translatable DNA sequences. A cistron is formed when a translatableDNA sequence is directionally inserted (directionally ligated) betweenthe control elements via the sequence of nucleotides adapted for thatpurpose. The resulting translatable DNA sequence, namely the insertedsequence, is, preferably, operatively linked in the appropriate readingframe.

DNA expression control sequences comprise a set of DNA expressionsignals for expressing a structural gene product and include both 5′ and3′ elements, as is well known, operatively linked to the cistron suchthat the cistron is able to express a structural gene product. The 5′control sequences define a promoter for initiating transcription and aribosome binding site operatively linked at the 5′ terminus of theupstream translatable DNA sequence.

Thus, a DNA expression vector of this invention provides a system forcloning translatable DNA sequences into the cassette portion of thevector to produce a cistron capable of expressing a fusion protein ofthis invention.

Cell Lines

A preferred cell line of the present invention is capable of continuousgrowth in culture and capable of expressing mammalian Class I MHCmolecules and assisting molecules on the surface of its cells. Any of avariety of transformed and non-transformed cells or cell lines areappropriate for this purpose, including bacterial, yeast, insect, andmammalian cell lines. (See, e.g., Current Protocols in MolecularBiology, John Wiley & Sons, NY (1991), for summaries and procedures forculturing and using a variety of cell lines, e.g., E. coli and S.cerevisiae.)

Preferably, the cell line is a eukaryotic cell line. More preferably,the cell line is poikilothermic (i.e., less sensitive to temperaturechallenge than mammalian cell lines). More preferably, it is an insectcell line. Various insect cell lines are available for use according tothe present invention, including moth (ATCC CCL 80), armyworm (ATCC CRL1711), mosquito larvae (ATCC lines CCL 125, CCL 126, CRL 1660, CRL 1591,CRL 6585, CRL 6586) and silkworm (ATCC CRL 8851). In a preferredembodiment, the cell line is a Drosophila cell line such as a Schneidercell line (see Schneider, J. Embryol. Exp. Morph. 27: 353-365 (1972));preferably, the cell line is a Schneider 2 (S2) cell line (S2/M3)adapted for growth in M3 medium (see Lindquist, et al., DrosophilaInformation Service 58: 163 (1982)).

Schneider cells may be prepared substantially as follows. Drosophilamelanogaster (Oregon-R) eggs are collected over about a 4 hour intervaland are dechlorinated in 2.5% aqueous sodium hypochlorite andsurface-sterilized by immersion in 70% ethanol for 20 minutes, followedby an additional 20 minutes in 0.05% HgCl.sub.2 in 70% ethanol. Afterbeing rinsed thoroughly in sterile distilled water, the eggs aretransferred to petri dishes containing sterile Metricel black filtersbacked with Millipore prefilters, both previously wetted with culturemedium. The eggs are placed overnight in a 22° C. incubator and removedfor culturing when 20-24 hours old. The embryos are each cut into halvesor thirds, then placed in 0.2% trypsin (1:250, Difco) in Rinaldini'ssalt solution (Rinaldini, Nature (London) 173: 1134-1135 (1954)) for20-45 minutes at room temperature. From 100-300 embryos are used toinitiate each culture.

After the addition of fetal bovine serum (FBS), the fragments arecentrifuged at 100×g for 2-3 minutes, resuspended in 1.25 ml culturemedium and seeded into glass T-9 flasks. The cultures are maintained atabout 22-27° C.+−.0.5° C., with a gaseous phase of ambient air.Schneider's culture medium (Schneider, J. Exp. Zool. 156: 91-104 (1964);Schneider, J. Embryol. Exp. Morph. 15: 271-279 (1966)) containing anadditional 500 mg bacteriological peptone per 100 ml medium andsupplemented with 15% inactivated FBS is preferably used. The pH(preferably 6.7-6.8) is monitored with 0.01% phenol red. The cell linesare preferably maintained by subculturing every 3-7 days. The cellsreadily attach to the glass but not so firmly as to require trypsintreatment; typically, simple pipetting is adequate to flush most of thecells from the bottom of the flasks. The morphological appearance of thecells is described in Schneider, J. Embryol. Exp. Morph. 27: 353-365(1972). They are essentially epithelial-like in appearance and rangefrom about 5-11 μm in diameter and 11-35 μm in length. Small pocketscontaining rounded cells may be dispersed randomly throughout the othercells.

Preferably, the Schneider 2 (S2) cells are maintained in Schneider'sDrosophila medium plus 10% FBS including penicillin (100 unit/ml) andstreptomycin (100 mg/ml). It is preferable to keep the cells at adensity of more than 0.5×10⁵/ml, and to grow them at a 24-30° C.temperature range. The cells tend to double in fewer than 24 hours andgrow to high cell density, i.e., about 2×10⁷/ml or greater. The cellsmay also be frozen in 90% FBS and 10% DMSO, for later use or analysis.One may place the cells at −70° C. and then store in liquid nitrogen.

A preferred cell line according to the present invention, identified asSchneider 2 (S2) cells, has been deposited pursuant to Budapest Treatyrequirements with the American Type Culture Collection (ATCC),Rockville, Md., on Feb. 18, 1992, and was assigned accession number CRL10974.

Cells of the present invention are transfected with cDNAs encoding(human) MHC heavy chains, β-2 microglobulin and one or more assistingmolecules, which have each been inserted into (i.e., operatively linkedto) an expression vector. In a more preferred embodiment, the vectorcomprises Drosophila expression plasmid pRmHa-3, into which expressiblenucleotide sequences encoding human Class I MHC heavy chains, human β-2microglobulin or human assisting molecules have been inserted usingtechniques disclosed herein. Preferably, the cDNAs encoding MHC heavychains, those encoding β-2 microglobulin and those encoding assistingmolecules are operatively linked to separate expression plasmids and arecotransfected into the cultured cells. Alternatively, the cDNAs encodingMHC heavy chains, β-2 microglobulin and assisting molecules may beoperatively linked to the same expression plasmid and cotransfected viathat same plasmid. In another variation, cDNAs encoding MHC heavychains, β-2 microglobulin, assisting molecules, and a cytokine such asIL-2 are operatively linked to expression plasmids and are cotransfectedinto a cell line of the present invention. Selection of HLA genes,construction of appropriate vectors and primer selection are describedin greater detail above.

Successfully transformed cells, i.e., cells that contain an expressiblehuman nucleotide sequence according to the present invention, can beidentified via well-known techniques. For example, cells resulting fromthe introduction of a cDNA or rDNA of the present invention can becloned to produce monoclonal colonies. Cells from those colonies can beharvested, lysed, and their DNA content examined for the presence of therDNA using a method such as that described by Southern, J. Mol. Biol.98: 503 (1975). In addition to directly assaying for the presence ofrDNA, successful transformation or transfection may be confirmed bywell-known immunological methods when the rDNA is capable of directingthe expression of a subject chimeric polypeptide. For example, cellssuccessfully transformed with an expression vector may produce proteinsdisplaying particular antigenic properties which are easily determinedusing the appropriate antibodies. In addition, successfultransformation/transfection may be ascertained via the use of anadditional vector bearing a marker sequence, such as neomycinresistance, as described hereinabove.

It is also preferable that the culture be stabile and capable ofsustained growth at reduced temperatures. For example, it is preferredthat the culture be maintained at about room temperature, e.g., about24-27° C. In other embodiments, the culture is maintained at highertemperatures, particularly during the process of activating CD8⁺ cells.It is thus preferred that a culture according to the present inventionbe capable of withstanding a temperature challenge of about 30° C. toabout 37° C. Addition of β-2 microglobulin to a culture stabilizes theClass I MHC to at least a 30° C. challenge; addition of β-2microglobulin and peptides results in greater thermostability at highertemperatures, i.e., at 37° C.

In order to prepare the culture for expression of empty—or morepreferably, peptide-loaded—MHC molecules, the culture may first requirestimulation, e.g., via CuSO₄ induction, for a predetermined period oftime. After a suitable induction period—e.g., about 12-48 hours,peptides may be added at a predetermined concentration (e.g., about 100μg/ml). Peptides may be prepared as discussed below. After a furtherincubation period—e.g., for about 12 hours at 27° C.—the culture isready for use in the activation of CD8⁺ cells. While this additionalincubation period may be shortened or perhaps omitted, the culture tendsto become increasingly stable to temperature challenge if it is allowedto incubate for a time prior to addition of resting or naive CD8⁺ cells.For example, cultures according to the present invention to whichpeptide has been added are capable of expressing significant amounts ofpeptide-loaded Class I MHC molecules even when incubated for extendedperiods of time at 37° C.

Nutrient media useful in the culturing of transformed host cells arewell known in the art and can be obtained from numerous commercialsources. In embodiments wherein the host cell is mammalian, a“serum-free” medium is preferably used.

Human β-2 Microglobulin and Assisting Molecules

In order to establish a cell line capable of producing therapeuticallyuseful amounts of surface-expressed human Class I MHC molecules, it ispreferable to cotransfect a cell line of the present invention with avector operably linked to a nucleotide sequence encoding β-2microglobulin in order to effect appropriate levels of expression ofhuman MHC molecules in the cell line. While the nucleotide sequenceencoding mammalian β-2 microglobulin such as mouse β-2 microglobulinincreases the stability of the human Class I MHC molecules expressed inthe cell lines of the present invention, it is preferable to cotransfectthe cell line with a vector operably linked to an expressible nucleotidesequence encoding a human β-2 microglobulin.

As discussed above, a preferred vector according to the presentinvention includes a nucleotide sequence encoding at least a portion ofa mammalian β-2 microglobulin molecule operatively linked to the vectorfor expression. The gene for the assisting molecules can be linked tothe same or another vector. It is also feasible to construct a vectorincluding nucleotide sequences encoding both a Class I MHC heavy chainand a β-2 microglobulin.

The sequencing and primers used for the assisting molecules arediscussed in more detail below. However, the protocols are similar.

A human β-2 microglobulin cDNA sequence has been published (see Suggs,et al., PNAS 78: 6613-17, 1981) and the sequence was used as a templatefor a polymerase chain reaction (PCR) using the following primers:

5′ primer:5′ GCTTGGATCCAGATCTACCATGTCTCGCTCCGTGGCCTTAGCTGTGCTCGCGCTACTCTC 3′ (SEQID NO 15) 3′ primer 5′ GGATCCGGATGGTTACATGTCGCGATCCCACTTAAC 3′ (SEQ IDNO 16)

The primers are used in a standard PCR reaction (see above andreferences cited therein). The reaction products are extracted withphenol, purified using a Geneclean kit (Bio 101, San Diego, Calif.),digested with Bam HI and cloned into the Bam HI site of pBS (Stratagene,La Jolla, Calif.). After verification of the sequence, this Bam HIfragment is cloned into the Bam HI site of an appropriate expressionvector. In the preferred embodiment, human β-2 microglobulin cDNA issynthesized and operably linked to expression vector pRmHa-3.

Peptides

Virtually all cellular proteins in addition to viral antigens arecapable of being used to generate relevant peptide fragments that serveas potential Class I MHC ligand. In most mammalian cells, then, anyparticular MHC peptide complex would represent only a small proportionof the total MHC encoded molecules found on the cell surface. Therefore,in order to produce surface-expressed human Class I MHC molecules thathave an increased capacity to specifically activate CD8⁺ cells, it ispreferable to isolate and load peptide fragments of appropriate size andantigenic characteristics onto Class I molecules.

The peptides of the present invention bind to Class I MHC molecules. Thebinding occurs under biological conditions which can be created in vivoas well as in vitro. The exact nature of the binding of the peptidesneed not be known for practice of the invention.

In a preferred embodiment, the peptides to be loaded onto the Class IMHC molecules are antigenic. It is also preferred that the peptides beof a uniform size, preferably 8-mers or 9-mers, and most preferably,8-mers. It is also preferable that the peptides prepared for loadingonto the MHC molecules be of a single species; i.e., that all peptidesloaded onto the MHC be identical in size and sequence. In this manner,it is possible to produce monoantigenic peptide-loaded MHC molecules.

Peptides may be presented to the cells via various means. Preferably,peptides are presented in a manner which allows them to enter anintracellular pool of peptides. For example, peptides may be presentedvia osmotic loading. Typically, peptides are added to the culturemedium. The peptides may be added to the culture in the form of anintact polypeptide or protein which is subsequently degraded viacellular processes, e.g., via enzymatic degradation. Alternatively, theintact polypeptide or protein may be degraded via some other means suchas chemical digestion (e.g. cyanogen bromide) or proteases (e.g.chymotrypsin) prior to its addition to the cell culture. In otherembodiments, the peptides are presented in smaller segments which may ormay not comprise epitopic amino acid sequences.

In a preferred embodiment, a sufficient amount of protein(s) orpeptide(s) is added to the cell culture to allow the Class I MHCmolecules to bind and subsequently present a large density of thepeptide—preferably, with the same kind of peptide attached to eachMHC—on the surface of human Class I MHC-expressing cells of the presentinvention. It is also preferred to allow the human Class I MHC heavychains and human β-2 microglobulin to bind—i.e., to formheterodimers—prior to presenting peptide to the MHC moleculesintracellularly.

In another embodiment of the invention, peptides are added totransfected cells of the present invention in order to enhance thethermostability of the MHC molecules expressed by the cells. As notedabove, peptides are preferably added to the culture medium. Antigenicpeptides that bind to the Class I molecules serve to thermostabilize theMHC molecules and also increase the cell surface expression. Cultureswith added peptides which bind to the MHC molecules are thussignificantly less susceptible to temperature challenge than cultureswithout added peptide.

In one embodiment of the present invention, antigenic peptides arepresented to the transformed/transfected cell line in various forms. Forexample, an entire protein or other antigenic polypeptide may bedegraded chemically or enzymatically, for example, and added to the cellline in this form. For example, a protein of interest is degraded withchymotrypsin and the resultant mixture of peptide “fragments” is addedto a transformed or transfected cell culture; these cells are thenallowed to “choose” the appropriate peptides (which are often smallerpeptides, preferably 8mers or 9mers) to load onto the Class I MHCmolecules. Alternatively, an entire protein or polypeptide sequence maybe cloned into an appropriate vector and inserted into a procaryoticcell, whereby the cell generates significant amounts of the antigenicpolypeptide which are then harvested, purified, and digested intopeptides which are then added to the transformed/transfected eukaryoticcell culture. The cells again would be allowed to “choose” the peptidesto load onto the expressed MHC.

Isolation of Resting or Precursor CD8⁺ Cells

Resting (or naive or precursor) CD8⁺ cells—i.e., T-cells that have notbeen activated to target a specific antigen—are preferably extractedfrom the patient prior to incubation of the CD8⁺ cells with thetransformed cultures of the present invention. It is also preferred thatprecursor CD8⁺ cells be harvested from a patient prior to the initiationof other treatment or therapy which may interfere with the CD8⁺ cells'ability to be specifically activated. For example, if one is intendingto treat an individual with a neoplasia or tumor, it is preferable toobtain a sample of cells and culture same prior to the initiation ofchemotherapy or radiation treatment.

Methods of extracting and culturing lymphocytes are well known. Forexample, U.S. Pat. No. 4,690,915 to Rosenberg describes a method ofobtaining large numbers of lymphocytes via lymphocytopheresis.Appropriate culturing conditions used are for mammalian cells, which aretypically carried out at 37° C.

Various methods are also available for separating out and/or enrichingcultures of precursor CD8⁺ cells. Some examples of general methods forcell separation include indirect binding of cells to specifically-coatedsurfaces. In another example, human peripheral blood lymphocytes (PBL),which include CD8⁺ cells, are isolated by Ficoll-Hypaque gradientcentrifugation (Pharmacia, Piscataway, N.J.). PBL lymphoblasts may beused immediately thereafter or may be stored in liquid nitrogen afterfreezing in FBS containing 10% DMSO (Sigma Chemical Co., St. Louis,Mo.), which conserves cell viability and lymphocyte functions.

Alternative methods of separating out and/or enriching cultures ofprecursor cells include both positive and negative selection procedures.For positive selection, after lymphocyte-enriched PBL populations areprepared from whole blood, sub-populations of CD8⁺ lymphocytes areisolated therefrom by affinity-based separation techniques directed atthe presence of the CD8 receptor antigen. These affinity-basedtechniques include flow microfluorimetry, includingfluorescence-activated cell sorting (FACS), cell adhesion, and likemethods. (See, e.g., Scher and Mage, in Fundamental Immunology, W. E.Paul, ed., pp. 767-780, River Press, NY (1984).) Affinity methods mayutilize anti-CD8 receptor antibodies as the source of affinity reagent.Alternatively, the natural ligand, or ligand analogs, of CD8 receptormay be used as the affinity reagent. Various anti-T-cell and anti-CD8monoclonal antibodies for use in these methods are generally availablefrom a variety of commercial sources, including the American TypeCulture Collection (Rockville, Md.) and Pharmingen (San Diego, Calif.).

Negative selection procedures are utilized to effect the removal ofnon-CD8 from the CD8⁺ population. This technique results in theenrichment of CD8⁺ cells from the T- and B-cell population ofleucophoresed patients. Depending upon the antigen designation,different antibodies may be appropriate. (For a discussion and review ofnomenclature, antigen designation, and assigned antibodies for humanleucocytes, including T-cells, see Knapp, et al., Immunology Today 10:253-258 (1989) and Janeway et al., Immunobiology, supra.) For example,monoclonal antibodies OKT4 (anti-CD4, ATCC No. CRL 8002) OKT 5 (ATCCNos. CRL 8013 and 8016), OKT 8 (anti-CD8, ATCC No. CRL 8014), and OKT 9(ATCC No. CRL 8021) are identified in the ATCC Catalogue of Cell Linesand Hybridomas (ATCC, Rockville, Md.) as being reactive with human Tlymphocytes, human T-cell subsets, and activated T-cells, respectively.Various other antibodies are available for identifying and isolatingT-cell species.

In a further embodiment, CD8⁺ cells can be isolated by combining bothnegative and positive selection procedures. (See, e.g. Cai and Sprent,J. Exp. Med. 179: 2005-2015 (1994)).

Preferably, the PBLs are then purified. For example, Ficoll gradientsmay be utilized for this purpose. The purified PBLs would then be mixedwith syngeneic Drosophila cells preincubated with the appropriateantigenic peptides.

In Vitro Activation of CD8⁺ Cells

In order to optimize the in vitro conditions for the generation ofspecific cytotoxic T-cells, the culture of antigen-presenting cells ismaintained in an appropriate medium. In the preferred embodiment, theantigen-presenting cells are Drosophila cells, which are preferablymaintained in serum-free medium (e.g. Excell 400).

Prior to incubation of the antigen-presenting cells with the cells to beactivated, e.g., precursor CD8⁺ cells, an amount of antigenic peptide isadded to the antigen-presenting cell culture, of sufficient quantity tobecome loaded onto the human Class I molecules to be expressed on thesurface of the antigen-presenting cells. According to the presentinvention, a sufficient amount of peptide is an amount that will allowabout 200 to about 500,000 and preferably about 200 to 1,000 or more,human Class I MHC molecules loaded with peptide to be expressed on thesurface of each antigen-presenting cell. Preferably, theantigen-presenting cells are incubated with >20 μg/ml peptide.

Resting or precursor CD8⁺ cells are then incubated in culture with theappropriate antigen-presenting cells for a time period sufficient toactivate and further enrich for a population of CD8⁺ cells. Preferably,the CD8⁺ cells shall thus be activated in an antigen-specific manner.The ratio of resting or precursor CD8⁺ (effector) cells toantigen-presenting cells may vary from individual to individual and mayfurther depend upon variables such as the amenability of an individual'slymphocytes to culturing conditions and the nature and severity of thedisease condition or other condition for which the within-describedtreatment modality is used. Preferably, however, thelymphocyte:antigen-presenting cell (e.g. Drosophila cell) ratio ispreferably in the range of about 30:1 to 300:1. For example, in oneembodiment, 3×10⁷ human PBL and 1×10⁶ live Drosophila cells were admixedand maintained in 20 ml of RPMI 1640 culture medium.

The effector/antigen-presenting culture may be maintained for as long atime as is necessary to activate and enrich for a population of atherapeutically useable or effective number of CD8⁺ cells. In generalterms, the optimum time is between about one and five days, with a“plateau”—i.e. a “maximum” specific CD8⁺ activation level—generallybeing observed after five days of culture. In one embodiment of thepresent invention, in vitro activation of CD8⁺ cells is detected withina brief period of time after transfection of a cell line. In oneembodiment, transient expression in a transfected cell line capable ofactivating CD8⁺ cells is detectable within 48 hours of transfection.This clearly indicates that either stable or transient cultures oftransformed cells expressing human Class I MHC molecules are effectivein activating CD8⁺ cells.

Preferably, the enrichment and concordant activation of CD8⁺ cells isoptimal within one week of exposure to antigen-presenting cells.Thereafter, in a preferred embodiment, the enriched and activated CD8⁺cells are further purified by isolation procedures including siterestriction, resetting with antibody-red blood cell preparations, columnchromatography and the like. Following the purification, the resultingCD8⁺ cell preparation is further expanded by maintenance in culture fora period of time to obtain a population of 10⁹ activated CD8⁺ cells.This period may vary depending on the replication time of the cells butmay generally be 14 days. Activation and expansion of CD8⁺ cells hasbeen described by Riddell et al., Curr. Opin. Immunol., 5: 484-491(1993).

Separation of CD8⁺ Cells from Drosophila Cells

Activated CD8⁺ cells may be effectively separated from the stimulator(e.g., Drosophila) cells using one of a variety of known methods. Forexample, monoclonal antibodies specific for the stimulator cells, forthe peptides loaded onto the stimulator cells, or for the CD8⁺ cells (ora segment thereof) may be utilized to bind their appropriatecomplementary ligand. Antibody-tagged cells may then be extracted fromthe stimulator-effector cell admixture via appropriate means, e.g., viawell-known immunoprecipitation or immunoassay

Administration of Activated CD8⁺ Cells

Effective, cytotoxic amounts of the activated CD8⁺ cells can varybetween in vitro and in vivo uses, as well as with the amount and typeof cells that are the ultimate target of these killer cells. The amountwill also vary depending on the condition of the patient and should bedetermined via consideration of all appropriate factors by thepractitioner. Preferably, however, about 1×10⁶ to about 1×10¹², morepreferably about 1×10⁸ to about 1×10¹¹, and even more preferably, about1×10⁹ to about 1×10¹⁰ activated CD8⁺ cells are utilized for adulthumans, compared to about 5×10⁶-5×10⁷ cells used in mice.

Preferably, as discussed above, the activated CD8⁺ cells are harvestedfrom the Drosophila cell culture prior to administration of the CD8⁺cells to the individual being treated. It is important to note, however,that unlike other present and proposed treatment modalities, the presentmethod uses a cell culture system (i.e., Drosophila cells) that are nottumorigenic. Therefore, if complete separation of Drosophila cells andactivated CD8⁺ cells is not achieved, there is no inherent danger knownto be associated with the administration of a small number of Drosophilacells, whereas administration of mammalian tumor-promoting cells may beextremely hazardous.

Methods of re-introducing cellular components are known in the art andinclude procedures such as those exemplified in U.S. Pat. No. 4,844,893to Honsik, et al. and U.S. Pat. No. 4,690,915 to Rosenberg. For example,administration of activated CD8⁺ cells via intravenous infusion isappropriate.

HLA Typing

As noted previously, HLA haplotypes/allotypes vary from individual toindividual and, while it is not essential to the practice of the presentinvention, it is often helpful to determine the individual's HLA type.The HLA type may be determined via standard typing procedures and thePBLs purified by Ficoll gradients. The purified PBLs would then be mixedwith syngeneic Drosophila cells preincubated with the appropriateantigenic peptides—e.g., in therapeutic applications relating to viralinfections, cancers, or malignancies, peptides derived from viral- orcancer-specific proteins.

Continuing to use viral or malignant conditions as an example, in thoseinstances in which specific peptides of a particular viral- orcancer-specific antigen have been characterized, the synthesizedpeptides encoding these epitopes will preferably be used. In cases inwhich the preferred antigenic peptides have not been preciselydetermined, protease digests of viral- or cancer-specific proteins maybe used. As a source for such antigen, cDNA encoding viral- orcancer-specific proteins is cloned into a bacterial expression plasmidand used to transform bacteria, e.g., via methods disclosed herein.

After HLA typing, if Drosophila cells expressing the preferred HLA arenot available, cDNAs encoding the preferred HLA may be cloned via use ofthe polymerase chain reaction. The primers disclosed in section B.1.above (SEQ ID NO 1 through SEQ ID NO 12) may be used to amplify theappropriate HLA-A, -B, -C, -E, -F, or -G cDNAs in separate reactionswhich may then be cloned and sequenced as described in the methodsdisclosed for HLA A2.1 below. Stable cell lines expressing the clonedHLA may then be established in the Drosophila cells. Alternatively, apopulation of insect cells transiently expressing a bulk population ofcloned recombinant molecules from the PCR reaction may be used for invitro CD8⁺ activation.

EXAMPLES

The following examples are intended to illustrate, but not limit, thepresent invention.

Example 1 Expression of Human Class I MHC Molecules

A. Preparation of pRmHa-3 Expression Vector

The pRmHa-3 expression vector for use in expressing MHC proteins inDrosophila Schneider 2 (S2) cells as described in this invention wasconstructed by ligating a Sph I linearized pRmHa-1 DNA expression vectorwith a DNA fragment resulting from a Sph I restriction digest of apRmHa-2 expression vector as described below. The ligating of pRmHa-1with the pRmHa-2 fragment in this manner was performed to remove one oftwo Eco RI restriction endonuclease cloning sites present in pRmHa-1.Thus, the resultant pRmHa-3 expression vector contained only one Eco RIrestriction site in the multiple cloning site (polylinker) into whichvarious MHC-encoding DNA fragments were inserted as described in theExamples.

1. Preparation of pRmHa-1 Expression Vector

The pRmHa-1 expression vector, containing a metallothionein promoter,metal response consensus sequences (designated MT) and an alcoholdehydrogenase (ADH) gene containing a polyadenylation signal isolatedfrom Drosophila melanogaster, was constructed as described by Bunch etal., Nucl. Acids Res. 16: 1043-61 (1988). A schematic of the finalpRmHa-1 construct is shown in FIG. 2. The plasmid expression vector,pUC18, having the ATCC accession number 37253, was used as the sourcevector from which subsequent vectors described herein were derived. ThepUC18 plasmid contains the following restriction sites from 5′ to 3′ inthe multiple cloning site, all of which are not illustrated in theschematic representations of the pUC18-derived vectors in FIG. 1: EcoRI; Sac I; Kpn I; Sma I and Sma I located at the same position; Bam HI;Xba I; Sal I, Acc I and Hinc II located at the same position; Pst I; SphI and Hind III. The pUC18 vector was first digested with Hind III toform a linearized pUC18. Blunt ends were then created by filling in theHind III ends with DNA polymerase I large fragment as described byManiatis et al., Molecular Cloning: A Laboratory Manual, eds. ColdSpring Harbor Laboratory, New York (1982).

The resultant linearized blunt-ended pUC18 vector was ligated with a 740base pair (bp) Hinf I fragment from the Drosophila melanogaster ADH genecontaining a polyadenylation signal. The ligated ADH allele was firstisolated from the plasmid PSACI, described by Goldberg et al., PNAS USA77: 5794-5798 (1980), by digestion with Hinf I followed by blunt endingwith Klenow resulting in the nucleotide sequence listed in SEQ ID NO 14.The pSACI vector containing the ADH allele was constructed by subcloninginto pBR322 (ATCC accession number 31344) a 4.7 kilobase (kb) Eco RIfragment of Drosophila DNA selected from a bacteriophage lambda librarycontaining random, high molecular weight (greater than 15 kb). The 5′Hinf I restriction site occurred naturally in the ADH gene at position1770 as described by Kreitman, Nature 304: 412-417 (1983). The 3′ Hinf Isite was derived from the pUC18 vector into which the ADH gene had beencloned. This position was four bases 3′ to the Xba I site at position2500 of the ADH gene. The ADH segment extended from the 35 bp upstreamof the polyadenylation/cleavage sequence in the 3′ untranslated portionof the ADH mRNA to 700 bp downstream of the polyadenylation signal. Theresultant pUC18-derived vector containing the ADH gene fragment wasdesignated pHA-1 as shown in FIG. 1.

The 421 bp Eco RI/Stu I MT gene fragment was obtained from a clonecontaining DNA of approximately 15.3 kb in a Drosophila melanogastergenomic DNA library. The library, prepared with a Mbo I partialdigestion of imaginal DNA, was cloned in the lambda derivative EMBL4.The fragment contained the MT promoter and metal response consensuselements of the Drosophila MT gene (Maroni et al., Genetics 112: 493-504(1986)). This region, containing the promoter and transcription startsite at nucleotide 1+, corresponded to position −370 to nucleotideposition +54 of the MT gene (SEQ ID NO 13). The resultant fragment wasthen ligated into pHA-1 expression vector prepared above that waspreviously linearized with Eco RI and Sma I. The 3′ blunt end in MTcreated by the Stu I digest was compatible with the blunt end in pHA-1created by the Sma I digest. The resultant pUC18-derived vectorcontaining a 5′ Drosophila MT gene fragment and a 3′ ADH gene fragmentwas designated pRmHa-1. The pRmHa-1 expression vector, shown in FIG. 2,contained the origin of replication (ori) and the beta-lactamase geneconferring resistance to ampicillin (Amp^(r)) from pUC18 as shown inFIG. 1 on the pHa-1 vector. The diagram of pRmHa-1 also shows the 5′ to3′ contiguous positions of the MT gene fragment, the multiple cloningsite and the ADH gene fragment. The pRmHa-1 vector was used as describedin c. below in the construction of the pRmHa-3 expression vector.

2. Preparation of pRmHa-2 Expression Vector

The construction of pRmHa-2 is shown in FIG. 1. For constructing thepRmHa-2 expression vector, the MT fragment prepared above was insertedinto the pUC18-derived vector pHA-1 as described for constructingpRmHa-1 above with a few modifications. An Eco RI linker was added tothe Stu I site of the Eco RI/Stu I-isolated MT gene fragment preparedabove to form a metallothionein fragment having Eco RI restriction siteson both ends. The resultant fragment was then ligated into the ADHfragment-containing pUC18 expression vector that was previouslylinearized with Eco RI. The resultant pUC18-derived vector containing a5′ Drosophila MT gene fragment and a 3′ ADH gene fragment having two EcoRI restriction sites 5′ to the multiple cloning site was designatedpRmHa-2. The pRmHa-2 expression vector, shown in FIG. 1, contained theorigin of replication (ori) and the beta-lactamase gene conferringresistance to ampicillin (Amp^(r)) from pUC18. The diagram of pRmHa-2also shows the 5′ to 3′ contiguous positions of the MT gene fragment,the multiple cloning site and the ADH gene fragment. The pRmHa-2 vectorwas used along with pRmHa-1 as described in c. below in the constructionof the pRmHa-3 expression vector.

3. Preparation of pRmHa-3 Expression Vector

To prepare the pRmHa-3 expression vector that had only one Eco RIrestriction site, a fragment from pRmHa-2 was ligated into pRmHa-1. Forthis construction, pRmHa-2, prepared in b. above, was first digestedwith Sph I. The resultant Sph I fragment beginning in the middle of theMT gene and extending to the Sph I site in the multiple cloning site wasfirst isolated from the pRmHa-2 vector and then ligated into pRmHa-1prepared in A.1. above. The pRmHa-1 vector-was previously modified toremove the Eco RI restriction site 5′ to the MT gene fragment thenlinearized with Sph I. This process is schematically illustrated in FIG.2. To remove the Eco RI site in pRmHa-1, the vector was first digestedwith Eco RI to form a linearized vector, then blunt ended with Mung Beannuclease and religated.

The pRmHa-1 vector lacking an Eco RI site was then digested with Sph Ito remove the region corresponding to the Sph I fragment insert frompRmHa-2 and form a linearized pRmHa-1 vector. The Sph I fragment frompRmHa-2 was then ligated into the Sph I linearized pRmHa-1 to form thepRmHa-3 expression vector. A schematic of the pRmHa-3 vector is shown inFIG. 3. The relative positions of the various restriction sites from thepUC18 vector from which pRmHa-3 was derived are indicated on the figure.In addition, the relative positions and lengths of the MT and ADH genefragments separated by the multiple cloning site (polylinker) into whichthe MHC gene of interest is cloned are indicated on the figure. ThepRmHa-3 vector, being derived from pUC18, contains the pUC18 origin ofreplication and beta-lactamase gene conferring ampicillin resistance.Thus, MHC encoding DNA fragments as prepared in this invention andcloned into the multiple cloning site of pRmHa-3 were transcriptionallyregulated by the MT promoter and polyadenylated via the ADH gene.

B. cDNA Synthesis

Detailed descriptions of the cDNA of Class I MHC molecules of variousHLA groups can be found in U.S. Pat. No. 5,314,813 to Peterson et al.which has been incorporated by reference.

cDNAs encoding any preferred HLA may be cloned via use of the polymerasechain reaction. The primers disclosed in section B.1. above (SEQ ID NO 1through SEQ ID NO 12) may be used to amplify the appropriate HLA-A, -B,-C, -E, -F, or -G cDNAs in separate reactions which may then be clonedand sequenced as described in the methods disclosed for HLA A2.1 above.Preparation of cDNA from human cells is carried out as described inEnnis, et al., PNAS USA 87: 2833-2837 (1990). Briefly, a blood sample isobtained from the individual and cells are collected aftercentrifugation and used to prepare total RNA. First strand cDNA issynthesized by using oligo(dT) and avian myeloblastosis virus reversetranscriptase. The resulting cDNA is used in a PCR amplificationreaction utilizing the appropriate primer(s) as noted in section B.1.above, and a GENEAMP kit and thermal cycler (Perkin-Elmer/Cetus).Reaction conditions are preferably as follows. 100 ng cDNA template and50 picomoles of each oligonucleotide primer are used. Thirty cycles arerun as follows: (a) one minute at 94° C.; (b) one minute at 60° C.; and(c) one minute, 30 seconds at 72° C. The PCR reaction is then heated to100° C. for 10 minutes to kill the Taq polymerase and the ends of theDNA made blunt by T4 polymerase (Stratagene, San Diego, Calif.).

To synthesize HLA A2.2, cDNA encoding a complete A2.2 (see Holmes, etal., J. Immunol. 139: 936-41 (1987), for the published sequence) iscloned into an M13 mp19 plasmid, a commercially available bacteriophagevector (Stratagene, La Jolla, Calif.). cDNA is synthesized by PCR usingprimers derived from the published sequence of A2. The cDNA is releasedfrom an M13 mp19 clone as a Not I (overhang filled with Klenow)/Eco RIfragment. (Klenow fragments are part of the E. coli DNA polymerase Imolecule, produced by the treatment of E. coli DNA pol I withsubtilisin. They are used to “fill out” 5′ or 3′ overhangs at the endsof DNA molecules produced by restriction nucleases.) The Not I/Eco RIfragment is inserted into pSP64T digested with Bg III (ends filled withKlenow) and Eco RI. pSP64T is an SP6 cloning vector designed to provide5′ and 3′ flanking regions from an mRNA which is efficiently translated(β-globin) to any cDNA which contains its own initiation codon. Thistranslation SP6 vector was constructed by digesting pSP64-Xβm with Bal Iand Bst EII, filling in the staggered ends with T4 DNA polymerase andadding a Bgl II linker by ligation. Bal I cuts the β-globin cDNA twobases upstream of the ATG (start codon) and Bst EII cuts eight basesupstream of the TAA (stop codon). There is only one Bg1 II site inpSP64T so that restriction enzymes cutting in the polylinker fragment,from Pst I to Eco RI can still be used to linearize the plasmid fortranscription. (See Kreig and Melton, Nucleic Acid Res. 12: 7057-7070,(1984), which also describes the construction of the plasmid pSP64-Xβm.)The resulting plasmid is cleaved with Eco RI (end filled with Klenow)and Hind III which is cloned into the pCMUII polylinker between Hind III(5′) and Stu I (3′). (See Paabo, et al., EMBO J. 5: 1921-1927 (1986).)The entire cDNA is removed as a Hind III (end filled with Klenow) Bam HIfragment which is cloned into pRmHa-3 cleaved with Sma I and Bam HI.

HLA A2.2 soluble form was prepared by engineering a stop codon into theabove-described A2.2 cDNA immediately preceding the transmembranedomain. The modification is achieved by cleaving the A2.2 cDNA cloned inthe eukaryotic expression vector PCMUII between Hind III 5′ and Stu I 3′(see above) with Mbo II and Bam HI inserting the followingoligonucleotides:

5′ primer: 5′ GGAGCCGTGACTGACTGAG 3′ (SEQ ID NO 17) 3′ primer:5′ CCCTCGGCACTGACTGACTCCTAG 3′ (SEQ ID NO 18)

The resulting recombinant plasmid is cleaved with Hind III, theoverhanging end filled with Klenow, then cut with Bam HI releasing arestriction fragment which is cloned into pRmHa-3 in the same way asA2.2 full length.

1. Construction of Murine ICAM-1 Expression Vector

Spleen cells were isolated from Balb/c mice. The spleen cells werestimulated with conA; mRNA was isolated using the FASTTRACK kit(Invitrogen, San Diego, Calif.) according to the manufacturers'instructions. cDNA was synthesized from the mRNA using AMV reversetranscriptase kit (Promega, Madison, Wis.) according to themanufacturers' instructions. Based on the published cDNA nucleotidesequence (Siu, G. et al., J. Immunol. 143, 3813-3820 (1989) thefollowing oligonucleotides were synthesized as PCR primers:

(SEQ ID NO 52) 5′: TTTAGAATTCAC CATGGCTTCA ACCCGTGCCA AG (SEQ ID NO 53)3′: TTTAGTCGACTC AGGGAGGTGG GGCTTGTCC

The cDNA synthesized was subjected to PCR using these primers. Theproduct was cleaved with the restriction enzymes Eco RI and Sal I andligated into pRmHa-3, which had been digested with the restrictionenzymes Eco RI and Sal I.

2. Construction of Murine B7.1 Expression Vector

Spleen calls were isolated from Balb/c mice and stimulated with conA.Messenger RNA was isolated using the FASTTRACK kit (Invitrogen, SanDiego, Calif.) according to the manufacturer's instructions. cDNA wassynthesized from the mRNA using AMV reverse transcriptase kit (Promega,Madison, Wis.) according to the manufacturer's instructions.

Based on the published cDNA nucleotide sequence (Freeman, et al., J.Exp. Med. 174: 625-631 (1991)) the following oligonucleotides weresynthesized as PCR primers:

(SEQ ID NO 54) 5′: TTTAGAATTCAC CATGGCTTGC AATTGTCAGT TG (SEQ ID NO 55)3′: TTTAGTCGACCT AAAGGAAGAC GGTCTGTTC

The cDNA synthesized was subjected to PCR using these primers. Theproduct was cleaved with the restriction enzymes Eco RI and Sal I andligated into pRmHa-3, which had been digested with the restrictionenzymes Eco RI and Sal I.

3. Construction of Murine B7.2 Expression Vector

IC-21 cells (obtained from ATCC) were propagated in RPMI 1640 mediumcontaining 10% Fetal Calf Serum. mRNA was isolated from these cellsusing the FASTTRACK kit (Invitrogen, San Diego, Calif.) according to themanufacturer's instructions. cDNA was synthesized from the mRNA usingAMV reverse transcriptase kit (Promega, Madison., Wis.) according to themanufacturer's instructions. Based on the published cDNA nucleotidesequence (Freeman, et al., J. Exp. Med. 178: 2185-2192 (1993)) thefollowing oligonucleotides were synthesized as PCR primers:

(SEQ ID NO 56) 5′: TTTAGAATTCAC CATGGACCCC AGATGCACCA TGGG (SEQ ID NO57) 3′: TTTAGTCGACTC ACTCTGCATT TGGTTTTGCT GA

The cDNA synthesized was subjected to PCR using these primers. Theproduct was cleaved with the restriction enzymes Eco RI and Sal I andligated into pRmHa-3, which had been digested with the restrictionenzymes Eco RI and Sal I.

The above expression constructs were transfected into Drosophila S2cells using the calcium phosphate method as listed in Table 1. Stablecell lines were selected by including 500 μg/ml Geneticin in the cellculture medium.

TABLE 1 MHC I B7.1 ICAM-1 Transfected (L^(d)) β2 (CD80) B7.2 (CD54)phsneo Cells μg μg μg μg μg μg 1 A 12 12 1 2 B 8 8 8 1 3 C 8 8 8 1 4 C 88 8 1 5 D 6 6 6 6 1 6 E 6 6 6 6 1 7 F 6 6 6 6 1 8 G 4.8 4.8 4.8 4.8 4.81

Human accessory and costimulatory molecules were cloned from human celllines demonstrated to express these proteins by FACS analysis withmonoclonal antibodies specific for the particular proteins. Adhesionmolecules belonging to the integrin family, ICAM-I (CD54) and LFA-3(CD58), were cloned from human cell lines K562 and HL60, respectively.The K562 cells, originated from human chronic myelogenous leukemia, wereobtained from ATCC(CCL-243) and cultured under conditions recommended(i.e., RPMI with 10% fetal calf serum at 37 degrees C. with 5% CO₂).HL60 cells, originated from a human promyelocytic leukemia, and wereobtained from ATCC(CCL-240) and were cultured according to ATCC'srecommendations. Costimulatory molecules B7.1 and B7.2 were also clonedfrom K562 and HL60 cells respectively.

4. cDNA

Messenger RNA samples were prepared from each cell line from RNAisolated by the modified guanidinium thiocyanate method (Chromczynski,et al. Anal. Biochem. 162: 156-159, 1987) followed by poly A+ RNAselection on oligo(dt)-cellulose columns (Sambrook, J., et al, MolecularCloning: A Laboratory Manual, Second Edition, 6.22-6.34, Cold SpringHarbor laboratory, CSH, NY), Induction of HL60 cells with vitamin D3(usually required to express some cell surface molecules) was notrequired to obtain the B7.2 and LFA-3 molecules, the proteins wereexpressed in the absence of induction. cDNA was prepared using AMVreverse transcriptase kit according to the manufacturers' instructions(Promega, Madison Wis.).

5. PCR Primers

PCR primers were designed and synthesized after obtaining copies of theknown sequences from the GENEWORKS database (Intelligenetics) andconsidering the ends needed to clone into the appropriate vectors. Theyare as follows with the top sequence of each protein the 5′ primer andthe bottom one the 3′ primer:

B7.1 5′-ACCCTTGAAT CCATGGGCCA CACACGGAGG CAG-3′ (SEQ ID NO 58)5′-ATTACCGGAT CCTTATACAG GGCGTACACT TTCCCTTCT-3′ (SEQ ID NO 59) B7.25′-ACCCTTGAGC TCATGGATCC CCAGTGCACT ATG-3′ (SEQ ID NO 60) 5′-ATTACCCCCGGGTTAAAAAC ATGTATCACT TTTGTCGCAT GA-3′ (SEQ ID NO 61) LFA-35′-ACCCTTGAGC TCATGGTTGC TGGGAGCGAC GCGGGG-3′ (SEQ ID NO 62)5′-ATTACCGGAT CCTTAAAGAA CATTCATATA CAGCACAATA CA-3′ (SEQ ID NO 63)ICAM-1 5′-ACCCTTGAAT TCATGGCTCC CAGCAGCCCC CGGCCC-3′ (SEQ ID NO 64)5′-ATTACCGGAT CCTCAGGGAG GCGTGGCTTG TGTGTTCGG-3′ (SEQ ID NO 65)

6. Expression of DNA Fragment

The cDNA preparations from each of the cell lines was used to clone thedesired proteins. The polymerase chain reaction was used to generatecDNA fragments utilizing the appropriate PCR primer (see above). Theappropriate DNA fragments were cloned into the Drosophila fly vectorpRMHA-3. Plasmid preparations have been prepared from all of thepreparations and are now ready for transfection into the fly cells.

Human β-2 microglobulin cDNA is prepared using a published partial cDNAsequence (see Suggs, et al., PNAS 78: 6613-17, 1981) is used as atemplate for a polymerase chain reaction (PCR) with the followingprimers:

5′ primer5′ GCTTGGATCCAGATCTACCATGTCTCGCTCCGTGGCCTTAGCTGTGCTCGCGCTACTCTC 3′ (SEQID NO 15) 3′ primer 5′ GGATCCGGATGGTTACATGTCGCGATCCCACTTAAC 3′ (SEQ IDNO 16)

The primers are used in a standard PCR reaction (see Nilsson, et al.,Cell 58: 707 (1989)). The reaction products are extracted with phenol,purified using a GENECLEAN kit (Bio 101, San Diego, Calif.), digestedwith Bam HI and cloned into the Bam HI site of pBS (Stratagene, LaJolla, Calif.). After verification of the sequence, this Bam HI fragmentis cloned into the Bam HI site of pRmHa-3.

As noted in the Examples, murine Class I cDNA was utilized in variousinstances. Murine Class I cDNA was prepared as follows.

H-2K^(b): cDNA encoding a complete K^(b) molecule is obtained from anexpression plasmid pCMU/K^(b) constructed as follows. A partial H-2K^(b)cDNA missing the leader sequence and most of the alpha I domain isprepared according to the method of Reyes, et al., PNAS 79: 3270-74(1982), producing pH 202. This cDNA is used to generate a full-lengthmolecule. The missing sequence is provided using a genomic cloneencoding H-2K^(b) (Caligan, et al., Nature 291: 35-39, 1981) as atemplate in a PCR reaction, using a 5′ primer flanked by a Not I site,followed by 21 nucleotides encoding the last seven amino acids of theleader sequence and 18 nucleotides complementary to the beginning of thealpha I domain and a 3′ primer complementary to the region encompassingthe Sty I site. The resulting fragment is ligated with pH 202 at the StyI site. The 5′ sequence encoding the remainder of the signal sequence isobtained form the D^(b) cDNA (see below) as a Bam HI/Not I fragment. Theentire coding sequence is cleaved from the expression plasmid as a BamHI fragment and cloned into pRmHa-3 cleaved with Bam HI.

H-2L^(d): cDNA encoding a complete L^(d) molecule is obtained from anexpression plasmid pCMUIV/L^(d) (see Joly and Oldstone, Gene 97: 213,1991). The complete cDNA is cleaved from a eukaryotic expression vectorPCMU IV/L^(d) as a Bam HI fragment and cloned into pRmHa-3 as K^(b).

As noted previously, the pCMU vector (pCMUIV) is derived from eukaryoticexpression vector pC81G as described in Nilsson, et al., supra. VectorpC81G, in turn, is derived from pA81G (Paabo, et al., Cell 33: 445-453(1983)) according to the method disclosed in Paabo, et al., EMBO J. 5:1921-7 (1986).

H-2D^(b): cDNA encoding a complete D^(b) molecule is obtained fromexpression plasmid pCMUIV/D^(b) (see Joly and Oldstone, Science 253:1283-85, 1991). The complete cDNA is cleaved from a eukaryoticexpression vector pCMUIV/D^(b) as a Bam HI fragment and cloned intopRmHa-3 as K^(b).

Murine β-2 microglobulin: full-length murine β-2 microglobulin cDNA isobtained as a Hind III (5′) (filled with Klenow)/Bgl II (3′) fragmentfrom pSV2neo (ATCC No. 37149) mouse β-2 microglobulin cDNA and clonedinto pRmHa-3 cleaved with Sma I and Bam HI.

Vector phshsneo confers neomycin (G418) resistance and is a derivativeof phsneo (pUChsneo) with an additional heat-shock promoter (hs)sequence, which may be synthesized from commercially-available pUC8 asdescribed in Steller, et al., EMBO J. 4: 167 (1985). The heat shockpromoter contained in these vectors is the hsp70 promoter. Other usefulvectors conferring neomycin resistance (G418 resistance) include cosmidvector smart2 (ATCC 37588), which is expressed under the control ofDrosophila hsp70 promoter, and plasmid vector pcopneo (ATCC 37409).

C. Insertion of Genes into Expression Vectors

The restriction products are subjected to electrophoresis on a 1%agarose gel (Maniatis, et al., Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory (1982)). The restriction fragmentsencoding the cDNAs are excised from the gel and purified away from theagarose using a GENECLEAN kit, according to manufacturers' directions(Bio 101, San Diego, Calif.). The expression plasmid pRmHa-3 (FIG. 3) iscleaved with the appropriate restriction enzymes in ONE PHOR ALL bufferaccording to the manufacturer's directions (Pharmacia, Piscataway, N.J.)and treated with alkaline phosphatase as described in the manufacturer'sliterature (Boehringer Mannheim, Indianapolis, Ind.). One hundred ng ofcleaved and phosphatased pRmHa-3 vector is mixed with 300 ng of agarosegel purified Class I MHC heavy chain cDNA or β-2 microglobulin cDNA andligated using T4 DNA ligase and ONE PHOR ALL buffer as described in themanufacturers' literature. After incubation at 16° C. for five hours,the ligation mixture is used to transform competent E. coli JM83(Maniatis, et al., supra (1982)).

Methods disclosed in Maniatis, et al., supra are used to prepare thecDNA needed. The presence of the MHC heavy chain cDNA and itsorientation in the vector is determined by restriction mapping. Bacteriacontaining the vector with the cDNA in the correct orientation relativeto the metallothionein promoter are used for large scale preparation ofDNA using the alkaline lysis method and cesium chloride gradientpurification. The amount of DNA obtained is determinedspectrophotometrically.

D. Transfection and Labeling of S2 Cells

S2 cells are grown in Schneider medium (Gibco/BRL, Grand Island, N.Y.)supplemented with 10% fetal calf serum (heat treated for one hour at 55°C.), 100 units/ml penicillin, 100 mg/ml streptomycin, and 1 mMglutamine. (For convenience, this supplemented medium is hereinafterreferred to as Schneider medium.) Cells are grown at 27° C. andtypically passaged every seven days by diluting 1:17 in fresh medium.Cells are converted to growth in serum free media (Excell 400 or 401supplemented with 100 units/ml penicillin, 100 mg/ml streptomycin, 1 mMglutamine, and 500 μg/ml G418 (JRH Biosciences, Lenexa, Kans.) byinitial dilution at 50% Schneider/50% Excell 401. One week later, cellsmay be passaged into 10% Schneider medium/90% Excell 401 and one weeklater into 100% Excell 401. Cells are maintained in this medium andpassaged every seven days by diluting 2:17 in fresh medium.

15×10⁶ S2 cells at a concentration of 10⁶ cells per ml are plated out in85 mm petri dishes. Twelve hours later, calcium phosphate/DNAprecipitates, prepared as described below (1 ml) are added dropwise tothe cells. After 48 hours, the supernatant is carefully removed and thecells transferred to a 175 cm² flask in a total volume of 50 ml inSchneider medium containing 500 μg/ml Geneticin (G418) (Gibco/BRL, GrandIsland, N.Y.). After 21 days, 20 ml of the culture is removed to a freshflask containing 30 ml of Schneider medium containing 500 μg/ml G418.Ten days later, a stable population of cells that weakly adhered to theflask and grew with a doubling time of approximately 24 hours isobtained and these cells are subsequently cultured and passaged in theselection media as described above. Frozen aliquots of these cells areprepared by collecting 5-20×10⁶ cells by centrifugation and resuspendingthem in 1 ml of cell freezing media (93% fetal calf serum/7%dimethylsulfoxide). Aliquots are then placed at −70° C. for one week andsubsequently transferred to liquid nitrogen storage.

Calcium phosphate precipitates are prepared as described by Paabo, etal. (EMBO J. 5: 1921-27 (1986)), except that 25 μg of DNA is used pertransfection. The following combinations of DNA are used to prepare theindicated transfectant:

(a) MHC Class I heavy chain alone: 23 μg heavy chain expression vectorDNA+2 μg of phshsneo DNA.

(b) MHC Class I heavy chain+β-2 microglobulin: 11.5 μg heavy chainexpression vector DNA+11.5 μg of β-2 microglobulin (human or mouse)expression vector DNA+2 μg of phshsneo DNA.

Other combinations of mouse genes are presented in Table 1.

Twenty-four hours prior to metabolic labeling, cells are plated out at acell density of 3-5×10⁶ cells/ml (10 ml/85 mm petri dish) in Schneidermedium containing 1 mM CuSO₄. Thirty minutes prior to labelling themedium is aspirated from the dishes and the cells are washed with 2×10ml of PBS and then incubated in Graces insect medium minus methionineand cysteine (special order from Gibco/BRL, Grand Island, N.Y.) for 20minutes, and then in 1 ml of this medium containing 0.1 mCi ³⁵S Translabel (New England Nuclear; dupont, Boston, Mass.). After the labellingperiod, the labelling solution is aspirated and the cells are eitherlysed immediately on ice, with ice cold PBS/1% Triton X100 (1 ml) orafter a chase period in the presence of methionine containing Schneideror Excell 400 medium (5 ml) (JRH Biosciences). The chase medium iscollected if soluble Class I MHC molecules are being analyzed.

The following operations are all carried out with the lysates kept cold(less than 8° C.). The lysates were collected into Eppendorf tubes,centrifuged in a microfuge tube for 15 minutes at 13,000×g, transferredto a fresh tube containing 100 μl of a 10% slurry of protein A sepharoseand placed on an end-over-end rotator for two hours. Following a furthercentrifugation in the microfuge for 15 minutes, the cell lysates areready for analysis.

In experiments utilizing murine MHC, S2 cells were transfected with themurine MHC recombinants described above using the CaPO₄ precipitationmethod; each heavy chain is transfected either alone or as a 50:50 mixwith the vector encoding β-2 microglobulin. A plasmid encoding neomycinresistance, phshsneo DNA, is included in each transfection such that apopulation of cells that stably expressed MHC Class I could be obtainedby growing the transfectants in selection medium (GeneticinG418-sulphate, Gibco/BRL, Grand Island, N.Y.).

E. Peptide Generation

Antigenic peptides according to the present invention may be obtainedfrom naturally-occurring sources or may be synthesized using knownmethods. In various examples disclosed herein, peptides are synthesizedon an Applied Biosystems synthesizer, ABI 431A (Foster City, Calif.) andsubsequently purified by HPLC.

Isolation or synthesis of “random” peptides may also be appropriate,particularly when one is attempting to ascertain a particular epitope inorder to load an empty MHC molecule with a peptide most likely tostimulate precursor CD8⁺ cells. One may produce a mixture of “random”peptides via use of proteasomes (see, e.g., Example 2.B.6) or bysubjecting a protein or polypeptide to a degradative process—e.g.,digestion with chymotrypsin—or peptides may be synthesized. While wehave observed that the cell lines of the present invention are able todegrade proteins and polypeptides into smaller peptides capable of beingloaded onto human Class I MHC molecules, it is preferable to introducesmaller peptides—e.g., 8-mers and 9-mers—directly into the cell cultureto facilitate a more rapid loading and expression process.

If one is synthesizing peptides, e.g., random 8-, 9- and 18-amino acidpeptides, all varieties of amino acids are preferably incorporatedduring each cycle of the synthesis. It should be noted, however, thatvarious parameters—e.g., solvent incompatibility of certain aminoacids—may result in a mixture which contains peptides lacking certainamino acids. The process should thus be adjusted as needed—i.e., byaltering solvents and reaction conditions—to produce the greatestvariety of peptides.

As noted hereinabove, murine heavy chains complexed with human β-2microglobulin were stable at temperatures approximately 6-8 degreeshigher than if complexed with murine β2. It was also observed that thestabilities imparted by peptide and xenogeneic β-2 microglobulin areadditive. A large increase in the thermostability of the Class Imolecules occurs if 8-9 mers are used, as compared to 12-25 mers;indeed, the difference between the stabilization imparted by the 8-9mers compared with the larger peptides might be even greater than whatwas observed previously, for even though the peptides have been purifiedby HPLC, it is likely that there is some contamination of the largerpeptides by 8-9 mers.

The thermostability of a Class I molecule is apparently dependent on:(1) the origin of β-2 microglobulin; (2) the presence of peptide; and(3) the length and sequence of this peptide.

Previous work (U.S. Pat. No. 5,314,813 to Peterson et al.; Jackson etal., PNAS USA 89: 12117-12121 (1992)) has shown that Class I MHC heavychains can bind peptide either alone or when they are associated withβ-2 microglobulin. Surface expression of peptide-loaded human Class IMHC, however, appears to be best facilitated by loading the moleculeswith peptide after the heavy chains have complexed with β-2microglobulin.

1. Expression of Human MHC

Once we determined that the thermostability of a Class I molecules isdependent on the origin of β-2 microglobulin, the presence of peptide,and the length and sequence of this peptide, we utilized thisinformation in the creation of cell lines capable of specificallyactivating CD8⁺ cells via the expression of peptide-loaded human Class IMHC molecules.

Thermolability appears to be an inherent property of Class I molecules;it has presumably evolved to ensure that Class I molecules which containeither no peptide or a peptide of poor binding properties (that conferslittle thermostability) self-destruct. In this way, the cell minimizesthe number of empty Class I molecules on its surface, for such asituation would presumably be dangerous in that exogenously derivedpeptides could be bound and presented. Human Class I molecules expressedin insect cells with human β2 are not stable to extended incubation at37° C.; neither are human Class I molecules expressed in the mutant cellline T2 which has been shown to be deficient in peptide loading onto theClass I molecules (Hosken and Bevan, Science 248: 367-70 (1990);Cerundolo, et al., Nature 345: 449-452 (1990)). Thus, it seems that theaffinity between the heavy chain and β-2 microglobulin has beencarefully conserved through co-evolution of the molecules such thatempty Class I molecules, or those carrying poorly-binding peptides,self-destruct at the body temperature of the “host” organism.

Human Class I MHC molecules were expressed in S2 cells. Cell linesco-expressing human β-2 microglobulin and HLA A2.2Y, HLA A2.1, HLA B7,or HLA B27 were established using previously-described methods. Briefly,cDNAs encoding the above proteins were cloned into the Drosophilaexpression vector pRmHa-3 and cotransfected with a human β-2microglobulin-containing plasmid and phshsneo plasmid into S2 cells viamethods disclosed herein. Three to four weeks later, the population ofG418-resistanT-cells was diluted 1:5 with fresh selection media. Once ahealthy growing population of cells was obtained, CUSO₄ was added to analiquot of cells and 24 hours later, cells were analyzed via flowcytometry using a monoclonal antibody W6/32 (ATCC HB95, Bethesda, Md.)which recognizes a monomorphic determinant of human Class I heavy chainswhen they are in association with β-2 microglobulin. (See Barnstable, etal., Cell 14: 9 (1978).) High levels of surface expression of each ofthe human Class I molecules were induced by the addition of CuSO₄ (datanot shown). These stable populations were sorted for high expressingcells using cytofluorimetry as described below. It is these sortedpopulations of cells which were used for all subsequent experiments.

Twenty-four hours prior to FACS analysis, CuSO₄ is added to the stablytransfected S2 cells (3-4×10⁶ cells/ml) to a final concentration of 1mM, thereby “switching on” expression from the transfected genes. Cellsare plated out in 24-well cluster dishes (2 ml per well). Eight hoursprior to FACS analysis, the CuSO₄ medium is replaced with fresh medium(1 ml) with or without peptide at a concentration of 50 μg/ml. 37° C.temperature challenges are carried out by transferring the dishes onto aflat surface in a 37° C. room at various time intervals prior toharvesting the cells for analysis.

To analyze surface expression of Class I MHC on the S2 cells, aliquotsof cells (5×10⁵) are transferred into tubes on ice, collected bycentrifugation (1,000×g for 4 minutes), resuspended in 3 ml of PBS/1%BSA, 0.02% sodium azide, collected by centrifugation and resuspended inPBS/BSA (0.5 ml) containing the appropriate primary antibody (ascitesfluids Y3, 28:14:8S, 30.5.7, W6/32, diluted 1:200). Rabbit antisera arediluted 1:500 and B22.293 hybridoma supernatant is used directly. Aftera one hour incubation on ice, cells are washed twice in 3 ml of PBS/BSAand resuspended in 0.5 ml of PBS/BSA containing FITC labeled secondaryantibody (Cappell, Durham, N.C.) and 1 ng/ml propidium iodide. After a30 minute incubation on ice, cells are washed once with PBS/BSA andresuspended in this buffer at a concentration of 1×10⁶/ml. Samples arethen analyzed by FACS 440 (Becton Dickinson). Dead cells stained withpropidium iodide, are excluded by including a live gate in the analysis.

For cell sorting, the same procedure outlined above is used, except thatall staining operations are carried out in a sterile hood. Solutions,including antibodies, are filter-sterilized, and Schneider media orExcell 400 is used in place of PBS/BSA. Cells that specifically boundthe primary antibody are sorted using a Becton Dickinson cell sorter.Sorted cells (2-8×10⁵) are washed once in medium before plating out at aconcentration of 2×10⁵ cells/ml.

F. Loading of Membrane-Bound Empty MHC Molecules by In Vitro Incubationwith Peptides

In order to demonstrate that the human Class I molecules expressed onthe surface of the Drosophila cells were empty, the cells were incubatedat 37° C. for two hours and the cell surface expression was analyzed bycytofluorimetry. The surface expression of both HLA B27 and A2.1 isgreatly reduced if cells are incubated at 37° C. for 2 hours; however,preincubating the cells in HIV peptides known to bind to the Class Imolecules affords significant thermal stability to the Class I, whilepeptides that do not bind have little effect (see FIG. 4). (A 9-aminoacid peptide ILKEPVHGV (SEQ ID NO 42) from the POL protein of HIV bindsand stabilizes HLA A2.1. A nine-amino-acid peptide from the Vpr proteinof HIV binds and stabilizes B27 (FRIGCRHSR; SEQ ID NO 41). These datashow that the human Class I molecules expressed on the surface ofDrosophila cells are empty and can be stabilized by binding specific HIVpeptides.

FIGS. 4 and 5 show peptide-induced thermostabilization of HLA B27 andHLA A2.1 expressed on the surface of Drosophila cells by HIV peptides.Drosophila cells expressing either HLA B27 or A2.1 were incubated withpeptides where indicated and then either maintained at 28° C. orincubated at 37° C. for two hours prior to analysis of the surfaceexpression of the Class I molecules by use of the antibody W6/32 (fromATCC HB95) and cytofluorimetry. The mean fluorescence of each cellpopulation is shown plotted against the incubation conditions The HIVPOL peptide (ILKEPVHGV, SEQ ID NO 42) stabilizes A2.1 but not B27 (FIG.4), while the HIV Vpr peptide (FRIGCRHSR, SEQ ID NO 41) stabilizes B27,but not A2.1 (FIG. 5).

Example 2 Preparation of Synthetic Antigen-Presenting Cells

A. Osmotic Loading

Osmotic loading of SC2 and 3T3 cells with ovalbumin protein was carriedout as described by Moore, et al., Cell 54: 777-785 (1988). The assayprocedure is as follows. In a 96-well dish, 1×10⁵ Drosophila cells (withor without peptide/protein loaded) or 3T3 cells were cocultured with1×10⁵ B3/CD8 T-cell hybridoma cells in 200 μl of RPMI media supplementedwith 10% fetal bovine serum. After 24 hours of incubation, 100 μl of thesupernatant from these cultures was added to 100 μl of RPMI containing5,000 CTLL cells. The cells were cocultured for 24 hours at 37° C. when1 μCi of ³H thymidine (Amersham) was added. After a further incubationof 15 hours at 37° C., the incorporation of radiolabel into the CTLLcells was determined by scintillation counting.

Assays conducted with murine MHC also verified that the insect cells arecapable of loading peptide onto the Class I molecules. Cells expressingas few as 200-500 MHC molecules containing a particular antigen can bedetected by a T-cell. As the Drosophila cells do not accumulatechromium, an antigen presentation assay based on B3/CD8, a T-cellhybridoma, was used. B3/CD8 is a hybridoma between B3, cytotoxic T-cellspecific for ovalbumin peptide 253-276 presented by H-2K^(b) Class Imolecules, and CD8-bearing IL-2-secreting cell line (see Carbone, etal., supra, 1989). Upon antigenic stimulation, B3/CD8 produces IL-2,measured by ³H thymidine incorporation in IL-2-dependent T-cell lineCTLL (Gillis, et al., J. Immunol. 120: 2027 91978)). Thus, by measuringthe amount of IL-2 produced, one can assay for T-cell recognition.

In order to provide an intracellular pool of ovalbumin protein fromwhich OVA peptides can be derived, ovalbumin (Sigma Chem. Co., MO) wasosmotically loaded into the cells as described by Moore, et al, supra(1988). Immediately after loading, the cells were mixed with the T-cellhybridoma. After two days' incubation, the medium was removed andassayed for IL-2. The amount of IL-2 was determined by the ability ofthe medium to support the growth of the IL-2-dependenT-cell line CTLL(Gillis, et al., supra, 1978), and growth was quantitated by the amountof radioactive thymidine incorporated into the cells.

S2 or 3T3 cells transfected with K^(b)/β2 were incubated with ovalbuminprotein (OvPro) or ovalbumin peptide, OVA 24 (OvPep) in isotonic (Iso)or hypertonic (Hyp) media. (Murine cell line BALB/3T3 is available fromthe ATCC under accession number CCL 163.) After treatment, cells werecocultured with the T-cell hybridoma B3/CD8. B3/CD8 is aT-cell-hybridoma between B3 (Carbone, et al., J. Exp. Med. 169: 603-12(1989)), cytotoxic T-cell specific for ovalbumin peptide 253-276presented by H-2K^(b) Class I molecules, and CD8-bearing IL-2-secretingcell line. Upon antigenic stimulation, B3/CD8 produces IL-2, measured by³H thymidine incorporation in IL-2-dependent cell line CTLL (Gillis, etal., J. Immunol. 120: 2027 91978)). Thus, by measuring the amount ofIL-2 produced, one can assay for T-cell recognition. The supernatantfrom the cocultures were analyzed for IL-2 by ³H thymidine incorporationby the IL-2-dependent cell line CTLL (ATCC No. TIB 214). The amount of³H thymidine incorporated is plotted against the initial celltreatments.

It can be seen in FIG. 6 that the T-cells responded well to theDrosophila cells if the ovalbumin peptide was added to the culturemedium, but no recognition occurred if the cells were loaded with theovalbumin protein. The MHC Class I molecules expressed on the cellsurface of the insect cell are fully functional in that they can bindpeptide if it is added to the culture medium and can present it in thecorrect context for it to be recognized by a T-cell.

B. Optimization of In Vitro Conditions

For the optimization of in vitro conditions for the generation ofspecific cytotoxic T-cells, the culture of Drosophila cell stimulatorcells is preferably maintained in serum-free medium (e.g. Excell 400).Drosophila cell stimulator cells are preferably incubated with >20 μg/mlpeptide. The effector:stimulator ratio (lymphocyte:Drosophila cellratio) is preferably in the range of about 30:1 to 300:1. The maximumspecific CD8⁺ is generally observed after five days of culture. Theculture of target cells for killing assay is preferably maintained in aserum-free medium.

Example 3 Stimulation of Proliferation and Differentiation of ArmedEffector T-Cells

We have found that Drosophila S2 cells transfected with MHC class Imolecules and specific assisting molecules are able to stimulate primaryresponses from T-cells in vitro. We present data below in this examplefrom a mouse model system. In this example, constructs coding for mouseMHC class I (L^(d)), β2 microglobulin, specific assisting molecules wereused and tested with CD8⁺ cells from lymph nodes of T-cell receptortransgenic mice.

The data in FIG. 7 provides evidence that the transfected Drosophila S2cells express the protein products of the transfected murine genes. Flowcytometry using a fluorescence-activated cell sorter (FACS) andfluorescently labeled antibodies were used to demonstrate the expressionof L^(d) (MHC molecule which includes heavy chain and β2) and thespecific assisting molecules B7.1 (CD80) and ICAM-1 (CD54) molecules bytransfected Drosophila S2 cells. Transfected cells were separated with aFACS to obtain cells expressing L^(d) molecules and were then maintainedin vitro.

The transfection of Drosophila S2 cells is summarized in Table 2. Thedata show L^(d), B7.1 and ICAM-1 expression measured by flow cytometryon the cell lines after induction with CuSO₄. It is apparent that,relative to the control antibody (ctr Ab), all of the transfectantsexpress L^(d) molecules on the cell surface. Likewise, cellscotransfected with L^(d) and B7.1 (L^(d).B7) express B7.1 but notICAM-1, whereas cells cotransfected with L^(d) and ICAM-1 (L^(d).ICAM)express ICAM-1 but not B7.1; triple transfection with L^(d), B7.1 andICAM-1 (L^(d).B7.1CAM) led to expression of all three molecules.

Using a standard tissue culture system (Cai, Z. and Sprent, J. (1994) J.Exp. Med. 179: 2005-2015), doses of 5×10⁴ purified CD8⁺ 2C lymph node(LN) cells were cultured at 37° C. with doses of 3×10⁵ transfected flycells±peptides (10 μM final concentration). Peptides were synthesized byR. W. Johnson Pharmaceutical Research Institute (Sykulev, et al. (1994)Immunity 1: 15-22. Proliferative responses were measured by adding ³HTdR(1 μCi/well) 8 hours prior to harvest. IL-2 production was measured byremoving supernatants from the cultures at 48 hours and adding 50 μlsupernatant to an IL-2 responsive indicator cell line (CTLL);proliferation of the indicator line was measured by addition of ³HTdR.The data shown in Table 2 are the means of triplicate cultures. Thetransfected Drosophila S2 cells die rapidly at 37° C. and fail toincorporate ³HTdR at this temperature.

The data in Table 2 demonstrate that the transfectants are able tostimulate primary responses of mouse T-cells.

Table 2. Capacity of transfected fly cells to stimulate primaryproliferative responses and IL-2 production by CD8⁺ lymph node cellsfrom 2C T-cell receptor transgenic mice.

TABLE 2 ³HTdR incorporation (cpm × 10³) with transfected fly cellsexpressing: L^(d) + L^(d) + B7.1 Peptides L^(d) + L^(d) + B7.1 +combined with Assay added L^(d) B7.1 ICAM-1 ICAM-1 L^(d) + ICAM-1Prolifer- — 0.2 0.1 0.3 0.2 — ation p2Ca 0.2 0.3 1.5 142.0 1.5 (Day 3)QL9 0.2 60.9 73.9 263.7 132.9 IL-2 — 0.3 0.2 0.1 1.2 — Production p2Ca0.2 0.2 0.1 64.6 0.3 (Day 2) QL9 0.1 0.4 0.2 158.6 0.5

The 2C T-cell receptor (TCR) is strongly reactive to L^(d) moleculescomplexed with certain peptides, e.g. p2Ca (SEQ ID NO 46) or QL9 (SEQ IDNO 47). These two peptides have moderate to high affinity for solubleL^(d) molecules, 4×10⁶ M⁻¹ for p2Ca, and 4×10⁹ M⁻¹ for QL9 (Sykulev. etal.). When complexed to soluble L^(d) molecules, the two peptides alsohave high binding affinity for soluble 2C TCR molecules. However, inboth TCR binding and L^(d) binding, the QL9 peptide clearly has a higheraffinity than the p2Ca peptide.

Table 2 shows that proliferative responses and IL-2 production by theresponder 2C cells to the weaker peptide, p2Ca, requires that thestimulator L^(d)-transfected cells coexpress both B7.1 and ICAM-1; amixture of cells expressing either L^(d)+B7.1 or L^(d)+ICAM-1 isnonstimulatory. By contrast, with the stronger peptide, QL9, L^(d).flycells expressing either B7 or ICAM elicit clearly-significant responses,although combined expression of B7 and ICAM generates much higherresponses. In contrast to these findings on T-cell proliferation, IL-2production in response to the QL9 peptide requires joint expression ofB7 and ICAM; expression of these molecules on separate cells isineffective.

The results show that Drosophila cells transfected with murine class Imolecules and costimulatory molecules induce murine T-cells to mountprimary proliferative responses and lymphokine (IL-2) production inresponse to peptide antigens. The system is also applicable to humanT-cells and could be used to stimulate unprimed (or primed) T-cellsspecific for tumor-specific antigens in vitro; in vivo infusion ofclonally-expanded T-cells specific for tumor-specific antigens might betherapeutic for patients with cancer. Infusion of T-cells specific forviral antigens would be useful in patients with viral infections, e.g.HIV.

Example 4 Immobilization of Biotinylated MHC Molecules on Avidin-CoatedRed Blood Cells

NHS-LC-biotin, neutravidin-and biotin-BMCC were purchased from Pierce(Rockford, Ill.). Sheep red blood cells were obtained from the ColoradoSerum Company (Denver, Colo.). Drosophila S2 cells expressing L^(d) andrecombinant L^(d) were prepared as described in Examples 1 and 2.Monoclonal antibodies 30.5.7 (anti-L^(d)) and 1B2 (anti-clonotypicantibody to the 2C T-cell receptor) were used as hybridoma cell culturesupernatants.

The protocol used is described by Muzykantov and Taylor (Anal. Biochem.(1994) 223, 142-148). Briefly, SRBC were washed 4 times in phosphatebuffered saline (PBS), biotinylated using NHS-LC-biotin, washed again 4times in PBS, incubated with neutavidin, and finally washed 4 times andstored at 4° C. in PBS containing 3% fetal calf serum and 0.02% sodiumazide.

Recombinant L^(d) was biotinylated using biotin-BMCC, amaleimide-coupled biotin which reacts with thiol groups. L^(d) displaysa free thiol group, the side chain of cystein 121, which is not in thepeptide binding site. Biotinylation was performed as recommended by themanufacturer. Unreacted biotin was removed using Centricon 10.

Biotinylated L^(d) was immobilized by incubation at a finalconcentration of 0.2 mg/ml with avidin-coated SRBC for 30 minutesfollowed by washing in DMEM containing 10% fetal calf serum. SRBC withattached L^(d) were used immediately.

T-cells expressing the 2C TCR transgene from lymph nodes of mice werepurified by magnetic depletion. Purified T-cells were consistently97-98% positive for staining in flow cytofluorometry using theanti-clonotypic antibody 1B2.

Immobilization of biotinylated L^(d) on avidin-coated SRBC was done asindicated above. Attachment was assessed using flow cytofluorometryusing anti-L^(d) antibody 30.5.7.

A typical experiment is represented in FIG. 8. The negative control(cells minus antibody) is shown in dotted lines. The filled peakcomprises cells labeled with fluorescent antibody. 99.78% of the cellswere labeled. Fluorescence intensity was in the same range than thehighest levels of intensity that we observed for L^(d) on syntheticantigen-presenting cells.

K^(b) (MHC molecule which includes heavy chain and β2) was alsobiotinylated using the same procedure. We could immobilize biotinylatedK^(b) on avidin-coated SRBC as assessed by flow cytofluorometry (FIG.9). 99.88% of the cells were labeled.

Rosetting experiments verified that the attached MHC moleculesinteracted functionally with T-cells. Drosophila S2 cells expressingL^(d), L^(d)-coated SRBC were incubated with QL9 peptide (0.02 mM) or anirrelevant peptide (MCMV, 0.02 mM) for 30 minutes on ice; 2C+ T-cellswere then added, the proportion being 10 2C+ T-cells for 1 Drosophila S2cell, or 10 SRBC for 1 2C+ T-cell; the mixture was pelleted and kept onice for at least 30 min. Cells were then carefully resuspended androsettes were counted, a rosette being a Drosophila S2 cell bound to atleast 3 2C+ T-cells, or a 2C+ T-cell bound to at least 3 SRBC. Rosetteswere observed in all cases. Typically, 30-40% of the lymphocytes wereincluded in rosettes when QL9 peptide was added. No rosette was observedin the presence of the irrelevant peptide, although occasionalattachment of a few single cells was observed.

These examples describe a new method to immobilize high amounts of MHCclass I molecules on various surfaces (fly cells, red blood cells, latexbeads) in native conformation as judged by monoclonal antibody bindingand resetting experiments (T-cell receptor binding). This method can beextended to other synthetic surfaces including artificial phospholipidmembranes. Phosphatidylethanolamine as well as avidin-coupledphospholipids are particularly relevant to our studies. Thesephospholipids are commercially available from Lipex Biomembrane Inc.,Vancouver, BC, Canada.

Example 5 Immobilization of Biotinylated MHC Molecules on Avidin-CoatedLatex Beads

Six micron diameter latex sulfate beads were purchased from InterfacialDynamics Corporation (Portland, Oreg.) and biotinylated according to theprotocol described in Example 4.

Avidin-coated latex beads were prepared using a 1% suspension of thelatex beads incubated in PBS containing 1 mg/ml of neutravidin for onehour at room temperature. An equal volume of PBS containing 10% fetalcalf serum was then added. After one hour of incubation at roomtemperature, the beads were washed 3 times and used for binding ofrecombinant biotinylated L^(d).

Recombinant biotinylated L^(d) was immobilized by incubation at a finalconcentration of 0.2 mg/ml with avidin-coated latex beads for 30 minutesfollowed by washing in DMEM containing 10% fetal calf serum. SRBC withattached L^(d) were used immediately.

Rosetting experiments verified that the attached MHC molecules on latexbeads interacted functionally with T-cells. Drosophila S2 cellsexpressing recombinant L^(d) and L^(d)-coated latex beads were incubatedwith QL9 peptide (0.02 mM) or an irrelevant peptide (MCMV, 0.02 mM) for30 minutes on ice; 2C+ T-cells were then added, the proportion being 102C+ T-cells for 1 Drosophila S2 cell, or L^(d)-coated latex beads for 12C+ T-cell; the mixture was pelleted and kept on ice for at least 30min. Cells were then carefully resuspended and rosettes were counted, arosette being a Drosophila S2 cell bound to at least 3 2C+ T-cells, or a2C+ T-cell bound to at least 3 latex beads. Rosettes were observed inall cases. Typically, 30-40% of the lymphocytes were included inrosettes when QL9 peptide was added. No rosette was observed in thepresence of the irrelevant peptide, although occasional attachment of afew single cells was observed.

Example 6 Immobilization and Detection of Recombinant Protein Bound toVarious Solid Supports Such as Plastic Microwell Plates

The MHC molecules were immobilized by direct binding to microtiterplates (Corning) and detected as follows:

MHC K^(b) molecules were diluted to desired concentration in PBS, e.g.0.001 mg/ml for 100 ng/well. 100 μl of diluted K^(b) was added to eachwell on the plastic microtiter plate. The plate was incubated for 1 hourat room temperature. After incubation, the plate was washed once withPBS and 200 μl 2% bovine serum albumin (BSA) in PBS+(0.05%) and Tween(PBST) was added, and incubated for another hour at room temperature.The plate was washed three times with PBST and biotinylated anti-K^(b)mAb was added (1:2500) in 2% BSA in PBS. The plate was incubated anotherhour at room temperature and washed three times with PBST. Avidinconjugated HRP was added (1:2500) in 2% BSA in PBS. Following anotherhour of incubation at room temperature, the plate was washed three timeswith PBST and H₂O₂ or thophenyldiamine was added. The reaction wasstopped with H₂SO₄. Reaction product was detected calorimetrically at490 nm.

FIG. 10 shows the results of detecting the presence of MHC K^(b)molecules using three different monoclonal antibodies.

Recombinant MHC K^(b) molecules can alternatively be bound throughbiotin-avidin linked interactions with the substrate. In thisembodiment, the microwell plates were coated with 100 μl avidin dilutedin PBS to a concentration of 0.001 mg/ml. Excess avidin was removed by aPBS wash. The above procedure for presenting and detecting K^(b) bindingfollowed.

Recombinant MHC molecules may alternatively be immobilized by a linkagebased on a poly-histidine tag added to the MHC interacting with thenickel bound to the substrate.

The above procedure for binding and detection is followed using nickelchelate coated microwell plates (Xenopore) and recombinant MHC moleculeswith a poly-histidine tag expressed using vector pRmHa/His₆ describedabove.

Example 7 Direct Binding of Peptide to Soluble Empty Class I MHCMolecules In Vitro

A. Procedures

H-2K^(b): prepared as described above in Example 1.B.

H-2K^(b) Sol: K^(b) sol cDNA is a derivative of K^(b), encoding theextracellular portion of the Class I MHC molecule. K^(b) sol cDNA may beproduced by PCR according to known methods, such as those described inEnnis, et al., PNAS USA 87: 2833-7 (1990) and Zemmour, et al.,Immunogenetics 33: 310-20 (1991). Specifically, cDNA encoding atruncated K^(b) molecule with a stop codon inserted at the end of thealpha 3 domain at amino acid position +275 is excised from the pCMUexpression plasmid as a Bam HI fragment and cloned into pRmHa-3 as K^(b)cDNA. The K^(b) sol cDNA is a derivative of the complete K^(b) cDNA (seeabove) which is used as a template in a PCR reaction using a 5′oligonucleotide that encompassed the Sty I site, and the following 3′oligonucleotide: 5′ ATATGGATCCTCACCATCTCAGGGTGAGGGGC 3′ (SEQ ID NO 43)

The resulting PCR fragment is blunt-end cloned into the Sma I site ofpBS (Stratagene, La Jolla, Calif.), sequenced, and the remaining 5′sequence of K^(b) cloned into the Sty I site. A cDNA encoding thecomplete K^(b)sol protein could be obtained as a Bam HI restrictionfragment.

H-2D^(b) and H-2L^(d) are prepared as discussed in Example 1.B. above.

The cDNAs encoding K^(b) α1α2α3 domains (274 residues) and murine β-2microglobulin (99 residues) were respectively cloned into the unique BamHI site of an expression vector harboring the metallothionein promoterpRMHa-3 (Bunch, et al., Nucleic Acid Res. 16: 1043-1061 (1988)).Drosophila S2/M3 cells were transformed with these recombinant plasmidsin addition to plasmid phshsneo (containing a neomycin-resistance gene)by the calcium-phosphate precipitation method described previously. Thetransformed cells selected against neomycin-analog antibiotics G418 weregrown at 27° C. in serum-free medium and soluble heavy-chain K^(b) andβ-2 microglobulin were co-expressed by the addition of 0.7 mM CuSO₄.

The soluble, assembled heterodimer of K^(b) was purified from theculture supernatants by affinity chromatography using anti-K^(b)monoclonal antibody Y3, followed by ion-exchange chromatography on aPharmacia Mono Q FPLC column according to the instructions of themanufacturer (Pharmacia, Piscataway, N.J.). SDS-polyacrylamide gelelectrophoresis (SDS-PAGE) of the K^(b) preparation followed by stainingwith Coomassie blue showed only one band of relative molecular mass (Mr)at about 32,000 and one band of Mr at about 12,000 with no detectableimpurities. The highly-purified K^(b) was dialyzed againstphosphate-buffered saline (PBS), filter-sterilized, and used for furtherstudy. Extinction coefficient of the soluble K^(b) (“K^(b)sol”) protein(43.2 kDa) is 69,200 M⁻¹ cm⁻¹ at 280 nm.

The purified K^(b) sol (0.3 μM) in PBS with or without 1% TX-100 wereexposed to varying temperatures (i.e., 4°, 23°, 32°, 37°, 42°, and 47°C.) for one hour. The proteins were then immunoprecipitated byincubating with the monoclonal antibody Y3 and protein A sepharose beads(Pharmacia, Piscataway, N.J.) at 4° C. for two hours, respectively. Thesamples were analyzed by 12.5% SDS-PAGE, followed by staining withCoomassie blue. The two thick bands on the gel are heavy and lightchains of antibody Y3. In another procedure, K^(b)sol (0.3 μM) wereincubated with 50 μM of peptides in PBS at 23° C. for two hours to allowfor K^(b)sol-peptide complex formation. After the addition of 1% TX-100,the samples were exposed to 12° C., 37° C., or 47° C. temperatures forone hour. The complexes were immunoprecipitated and analyzed by SDS-PAGEas described above. In a third procedure, K^(b)sol (2.7 μM) wereincubated with 50 μM of OVA-8, VSV-8 or SEV-9 peptides, respectively, at23° C. for two hours. The samples were applied on a 5% polyacrylamideIEF gel. IEF was run from pH 5-7 and the gel was stained with silver.

Next, VSV-8 peptide was radioiodinated using the chloramine-T method(Hunter, et al., Nature 194: 495-6 (1962)) and free ¹²⁵I was removed byC₁₈ column (OPC cartridge, Applied Biosystems, Foster City, Calif.). Thelabeled peptide was further purified by C₁₈ reverse-phase HPLC. Afterelution, the labeled peptide was lyophilized and resuspended in PBS.

The specific activity of [¹²⁵I]VSV-8 (about 250 Ci/mmole) was determinedspectrophotometrically by using extinction coefficient of tyrosine at274 nm (1420 M⁻¹ cm⁻¹). First, K^(b)sol (0.5 μM) was mixed with[¹²⁵I]VSV-8 (1.5 nM) and unlabeled VSV-8 (50 nM) at 23° C. for 16 hoursto allow for complex formation. A portion of the sample was analyzed bygel filtration (Superose 12, Pharmacia, Piscataway, N.J.) in PBS. Afterelution, radioactivity contained in each fraction (0.05 ml) wasmeasured. Protein was monitored by absorbance at 280 nm.

In a second procedure, [¹²⁵I]VSV-8 (0.39 nM) was mixed with variousconcentrations of K^(b)sol in PBS containing 1% bovine serum albumin(BSA). After incubation at 23° C. for 2-16 hours, K^(b)sol-peptidecomplexes were separated from free peptide by small gel filtration(Bio-Gel P30, BioRad, Richmond, Calif.) in PBS. P30 gel filtrationpermitted over 95% separation of bound and free peptide within about 5minutes. Radioactivity of bound and free peptides was measured and thedata were analyzed by linear regression. At maximal levels of K^(b)soloffered, about 65% of the total labeled peptides were bound. Thismaximal binding capacity of labeled peptide to K^(b)sol proteindeteriorated over time, presumably due to radiation by ¹²⁵I bound toVSV-8.

In a third procedure, each sample contained 0.39 nM of [¹²⁵I]VSV-8(about 18,000 cpm), unlabeled peptides at the indicated concentration,and 30 nM of K^(b)sol that gives about 50% of the [¹²⁵I]VSV-8 binding inthe absence of unlabeled peptide at a final volume of 72 μl. Allcomponents were dissolved and diluted in PBS containing 1% BSA. Afterincubation for 2-16 hours at 23° C., 50 μl samples were analyzed by P30gel filtration as described above. The dissociation constants forunlabeled peptides were determined from molar concentrations of[¹²⁵I]VSV-8 and unlabeled peptides giving 50% inhibition of [¹²⁵I]VSV-8binding to K^(b)sol as described. (See Muller, et al., Meth. Enzymol.92: 589-601 (1983).)

K^(b)sol (0.3 μM) and [¹²⁵I]VSV-8 (0.39 nM) were then incubated at 4°C., 23° C., and 37° C., and the association was determined at varioustimes by P30 gel filtration. Murine β-2 microglobulin was added, whennecessary, before the incubation at the indicated concentration. Themurine β-2 microglobulin was prepared by affinity chromatography usinganti-β-2 microglobulin polyclonal antibody K355 from culturesupernatants of the recombinant Drosophila cells. (See also Logdberg, etal., Molec. Immun. 14: 577-587 (1979).) In another experiment, K^(b)sol(0.3 μM or 1.8 μM) and [¹²⁵I]VSV-8 (2.4 nM) were incubated at 23° C. fortwo hours, and the peptide-K^(b)sol complexes were isolated by P30 gelfiltration. The samples contained very small amounts of [¹²⁵I]VSV-8 andK^(b)sol complexes (at the maximum, 2.4 nM) and empty K^(b)sol at finalconcentration of about 50 to 300 nM. To some samples, 3 μM of β-2microglobulin, 3 μM of β-2 microglobulin plus 20 μM of unlabeled VSV-8,20 μM of unlabeled VSV-8, or 1% TX-100 were added. The samples wereincubated for various times at 37° C. and the degree of dissociation wasdetermined by passage over P30 columns.

B. Discussion

Class I MHC molecules present antigenic peptides to cytotoxic Tlymphocytes. Direct binding of peptide to Class I molecules in vitro hasbeen hampered by either the presence of previously bound peptides at thebinding site (Chen and Perham, Nature 337: 743-5 (1989)) or the lack ofbinding specificity. (See, e.g., Frelinger, et al., J. Exp. Med. 172:827-34 (1990); Choppin, et al., J. Exp. Med. 172: 889-99 (1990); Chen,et al., J. Exp. Med. 172: 931-6 (1990).) In vitro analysis of peptidebinding to soluble, empty Class I molecules purified from Drosophilacells transformed with truncated H-2K^(b)sol and murine β-2microglobulin genes is disclosed herein. The results demonstrate thatpeptide binding is very rapid and naturally processed peptides(octapeptides; see, e.g., Van Bleek, et al., Nature 348: 213-6 (1990);Falk, et al., Nature 351: 290-6 (1991)) have the highest affinities toK^(b)sol of the nanomolar range and indicate that K^(b)sol complexedwith octapeptides are stable, whereas those complexed with slightlyshorter or longer peptides are short-lived. Interactions between freeheavy chain and β-2 microglobulin is basically reversible in the absenceof detergent. Peptides spontaneously bind to empty Class I moleculeswithout dissolution of β-2 microglobulin. However, excess β-2microglobulin apparently promotes the binding of peptide to empty ClassI as a consequence of reassociation of free heavy chain with β-2microglobulin under conditions where the heterodimers are unstable.

Soluble H-2K^(b) molecules (composed of the α1α2α3 domain of heavychain) and murine β-2 microglobulin, were purified from the culturesupernatants of Drosophila cells which were concomitantly transformedwith the truncated heavy chain and β-2 microglobulin genes. Preliminaryexaminations suggested that Drosophila cells express Class I MHCmolecules devoid of endogenous peptides on the cell surface. Some of theproperties of empty Class I molecules include the observation that theyare less stable at 37° C. and their structure is stabilized by thebinding of peptide. (See, e.g., Schumacher, et al., Cell 62: 563-7(1990); Ljunggren, et al., Nature 346: 476-80 (1990).) To confirm thatpurified soluble K^(b) are also empty, their thermal stability indetergent-free solution was examined. Surprisingly, the proteins heatedfor one hour at 47° C. were well recovered by immunoprecipitation usinga conformational antibody, Y3. This unexpected result led us to adddetergent, 1% Triton X-100 (polyoxyethylene (9) octyl phenyl ether), tothe protein solution, since similar experiments to test the stability ofClass I molecules have always been conducted in detergent lysates (SeeSchumacher, et al., cited supra). The results obtained in the presenceof detergent show that the purified K^(b)sol is now unstable at 37° C.This and other lines of evidence suggest that K^(b)sol heterodimerdisassembles into the heavy chain and β-2 microglobulin at elevatedtemperatures and that detergent may prevent β-2 microglobulin fromreassociating with dissociated free heavy chain (see below). Second, thepossibility of stabilizing purified K^(b)sol with peptides was studied.The results of the first-described examination demonstrated that theproteins can be stabilized only when they are mixed with octapeptide(vesicular stomatitis virus nucleocapsid protein [VSV-8], see Table 3below) which is shown to be a naturally processed peptide (see VanBleek, et al., cited supra). These observations are consistent with thecharacteristics of empty Class I molecules mentioned above.

Independent support that the purified K^(b)sol molecules are empty isprovided by isoelectric focusing (IEF) under native conditions (data notshown). The soluble K^(b) purified from Drosophila cells exhibited amuch simpler pattern than HLA-A2 molecules purified from humanlymphoblastoid cell lines (see FIG. 3 in Silver, et al., Nature 350:619-22 (1991)). The complicated pattern of HLA-A2 on IEF is presumed tobe the result of the presence of heterogeneous peptides bound to themolecules. The simple band of purified K^(b)sol indicates the absence ofendogenous peptides. In addition, the incubation of K^(b)sol withantigenic peptides caused the distinct shifts of band on IEF gel,reflecting the change in isoelectric point of K^(b)sol due to thepeptide binding. It should be noted that such band-shifting was notobserved in HLA-A2 molecules when they were simply mixed with peptides,unless HLA-A2 are incubated with peptides in “reconstituting conditions”after removal of previously bound endogenous peptides. Taken together,these observations on native IEF also indicate that soluble K^(b)purified from Drosophila cells are empty.

The association of ¹²⁵I-labeled VSV-8 with K^(b)sol was demonstrated bygel filtration (not shown). The radioactivity of high molecular weightmaterials corresponds to peptide-K^(b)sol complexes, while that of lowmolecular weight materials represents free peptides. Unlabeled VSV andovalbumin (OVA) peptides could compete with the labeled VSV-8 (seebelow), arguing that [¹²⁵I]VSV-8 is bound specifically to K^(b)solmolecules. Reversed-phase HPLC revealed that K^(b)-bound [¹²⁵I]VSV-8 hasthe identical retention time to the input peptide. The binding toK^(b)sol of the labeled VSV-8 was saturable, exhibiting a dissociationconstant (K_(D)) of about 33 nM (not shown). From the x-axis of theScatchard plot, it was noted that about 65% of the labeled VSV-8 is ableto bind to K^(b).

To determine affinities of various peptides to K^(b), competitiveradioimmunoassays (RIA) using [¹²⁵I]VSV-8 were carried out (data notshown). The inhibitory peptides used for the RIA are listed in Table 3.K_(D) for each peptide is summarized in Table 3 as well.

TABLE 3 Various Antigenic Peptides* Used in Present Studies CodeSequence K_(D) (M) VSV-7 GYVYQGL 5.3 × 10⁻⁸ (SEQ ID NO 40, residues4-10) VSV-8 RGYVYQGL 3.7 × 10⁻⁹ (SEQ ID NO 40, residues 3-10) VSV-9NLRGYVYQGL 7.3 × 10⁻⁹ (SEQ ID NO 40, residues 2-10) VSV-10N DLRGYVYQGL3.9 × 10⁻⁷ (SEQ ID NO 40) VSV-9C RGYVYQGLK 6.9 × 10⁻⁹ (SEQ ID NO 44,residues 1-9) VSV-10C RGYVYQGLKS 2.1 × 10⁻⁸ (SEQ ID NO 44) OVA-8SIINFEKL 4.1 × 10⁻⁹ (SEQ ID NO 39, residues 5-12) OVA-9N ESIINFEKL 8.9× 10⁻⁸ (SEQ ID NO 39, residues 2-10) OVA-10N LESIINFEKL 2.8 × 10⁻⁷ (SEQID NO 39, residues 3-12) OVA-9C SIINFEKLT 1.1 × 10⁻⁸ (SEQ ID NO 39,residues 5-13) OVA-10C SIINFEKLTE 1.4 × 10⁻⁸ (SEQ ID NO 39, residues5-14) OVA-24 EQLESIINFEKLTEWTSSNVMEER 7.1 × 10⁻⁵ (SEQ ID NO 39) SEV-9FAPGNYPAL 2.7 × 10⁻⁹ (SEQ ID NO 45) VSV-8: Vesicular stomatitis virusnucleocapsid pro- tein 52-59 (Van Bleek, et al., Nature 348: 213-216(1990)) OVA-8: Ovalbumin 257-264 (Carbone, et al., J. Exp. Med. 169:603-12 (1989)); SEV-9: Sendai virus nucleoprotein 324-332 (Schumacher,et al., Nature 350: 703-706 (1991)) *All peptides were purified by C₁₈reversed-phase HPLC to exclude contaminating shorter peptides withdifferent binding properties. The 3-letter code designations and SEQ IDNO for each peptide are given below. VSV-7 GlyTyrValTyrGlnGlyLeu (SEQ IDNO 40, residue nos. 4-10) VSV-8 ArgGlyTyrValTyrGlnGlyLeu (SEQ ID NO 40,residue nos. 3-10) VSV-9N LeuArgGlyTyrValTyrGlnGlyLeu (SEQ ID NO 40,residue nos. 2-10) VSV-10N AspLeuArgGlyTyrValTyrGlnGlyLeu (SEQ ID NO 40)VSV-9C ArgGlyTyrValTyrGlnGlyLeuLys (SEQ ID NO 44, residue nos. 1-9)VSV-10C ArgGlyTyrValTyrGlnGlyLeuLysSer (SEQ ID NO 44) OVA-8SerIleIleAsnPheGluLysLeu (SEQ ID NO 39, residue nos. 5-12) OVA-9NGluSerIleIleAsnPheGluLysLeu (SEQ ID NO 39, residue nos. 4-12) OVA-10NLeuGluSerIleIleAsnPheGluLysLeu (SEQ ID NO 39, residue nos. 3-12) OVA-9CSerIleIleAsnPheGluLysLeuThr (SEQ ID NO 39, residue nos. 5-13) OVA-10CSerIleIleAsnPheGluLysLeuThrGlu (SEQ ID NO 39, residue nos. 5-14) OVA-24GluGlnLeuGluSerIleIleAsnPheGluLysLeuThrGlu-TrpThrSerSerAsnValMetGluGluArg (SEQ ID NO 39) SEV-9PheAlaProGlyAsnTyrProAlaLeu (SEQ ID NO 45)

The peptides of naturally processed size (8mer for VSV and OVA, and 9merfor sendai virus nucleoprotein [SEV]) had the highest and remarkablysimilar affinities from the range of 2.7 to 4.1 nM. this exceedinglyhigh affinity of the natural peptides is consistent with recentobservations. (See, e.g., Schumacher, et al., Nature 350: 703-6 (1991);Christnick, et al., Nature 352: 67-70 (1991).) However, peptides thatwere shorter or longer by as little as one or two residues lowered theaffinity by a factor of from 2 to 100. This reduction of the affinity iseven more drastic for a much longer peptide; i.e., the affinity of 24merpeptide (OVA-24) is more than 10,000-fold lower than that of OVA-8.These results help to explain why earlier reports using longer peptidesclaim the affinity of micromolar range. (See, e.g., Frelinger, et al.and Choppin, et al., both cited supra.) It is of particular interestthat the extension of peptides at the carboxyl terminus is much lessdestructive of the affinity than extension at the amino terminus.According to the three-dimensional structure of HLA-A2, thepeptide-binding groove is formed by two long α helices on theantiparallel β strands, and the cleft is about 25 angstroms long, whichis proposed to accommodate an extended peptide chain of about eightresidues (see, e.g., Bjorkman, et al., Nature 329: 506-12 (1987)). Atone end of the cleft, the α1 and α2 helices come close together tightly,while at the other end, the cleft is fairly open. It is now speculatedthat both VSV and OVA peptide bind to the cleft in the same orientation*and the carboxyl terminus of the peptides might interact with therelatively open end of the cleft so that the extension of peptide at thecarboxyl terminus does not cause severe steric hindrance.

Examinations were then performed to determine the rate of peptidebinding to K^(b) at 4° C. and 23° C., respectively (not shown). Bindingwas very rapid, especially at 23° C., with a half-time of about 5minutes even in extremely low concentrations of labeled peptides (about0.4 nM). This contrasts with previous observations, which show ahalf-time of association of about two hours. (See, e.g., Choppin, etal., cited supra.) Again, only 65% of the total labeled peptide was ableto bind. The addition of excess β-2 microglobulin did not affect thepeptide-binding kinetics at such low temperatures that K^(b) heterodimeris stable (remained to be assembled). This implies that exchange of β-2microglobulin is not a prerequisite for peptide binding; i.e., peptidescan spontaneously bind to empty Class I molecules without dissociationof β-2 microglobulin. In contrast, excess free β-2 microglobulinapparently promotes peptide binding at 37° C. (data not shown). As theconcentration of added β-2 microglobulin increased, more peptides boundto K^(b) molecules. Since empty K^(b) are unstable at 37° C., a fractionof heterodimers must be dissociated to the heavy chain and β-2microglobulin and thereby, the heterodimer must be in equilibrium withfree heavy chain and free β-2 microglobulin. Then, the addition of β-2microglobulin should shift the equilibrium toward the formation ofheterodimer that can bind peptides. This view is supported by recentobservations that there are substantial numbers of Class I free heavychains on the normal cell surface and exogenously added β-2microglobulin facilitates peptide binding to empty Class I molecules oncells as a consequence of the reassociation of β-2 microglobulin withfree heavy chain. (See, e.g., Rock, et al., Cell 65: 611-620 (1991);Kozlowski, et al., Nature 349: 74-77 (1991); Vitiello, et al., Science250: 1423-6 (1990).)

The dissociation kinetics of peptide at 37° C. were then observed.Immediately after isolating [¹²⁵I]VSV-8 and K^(b) complexes by gelfiltration, the samples containing either 50 or 300 nM K^(b) wereexposed to 37° C. temperatures. Some samples were supplemented with 3 μMβ-2 microglobulin and/or 20 μM unlabeled VSV-8, or 1% TX-100. Thedissociation of labeled peptides from K^(b) was measured at varioustimes (not shown). In the presence of a large excess of unlabeledpeptides, the dissociation rate of peptide followed first-order kineticswith a half-time dissociation of about 36 minutes (a dissociation rateconstant of 3.2×10⁻⁴ s⁻¹). This unexpected, relatively rapiddissociation of labeled peptide does not agree with some current viewsof stable peptide-Class I complexes. In fact, the results ascertained(not shown) demonstrated that K^(b) and VSV-8 complexes are stable. Thisdiscrepancy must arise from the 10-fold lower affinity of radiolabeledVSV-8 (33 nM) compared with that of unlabeled VSV-8 (3.7 nM).

The first-order kinetics were also observed when the detergent was addedinstead of unlabeled peptide, indicating that the detergent makes thepeptide dissociation process irreversible. In contrast, the peptidedissociation profile did not follow the first-order kinetics in theabsence of unlabeled peptide or the detergent. This suggests that thepeptide association/dissociation is reversible and the binding ofpeptide is dependent on the concentration of heterodimer (compare thekinetics between 50 nM and 300 nM of K^(b)). This became more evidentwhen excess β-2 microglobulin was added. These results support theprevious argument that interaction between the heavy chain, β-2microglobulin and peptide are basically reversible at 37° C., if notentirely, in the absence of detergent. It is probable that a detergentsuch as TX-100 may prevent β-2 microglobulin from reassociating withfree heavy chain at 37° C. This could reasonably explain why K^(b) onceheated to elevated temperatures in the absence of detergent can beefficiently immunoprecipitated by conformational antibody (not shown).Interestingly, the addition of β-2 microglobulin did not suppress thepeptide dissociation in the presence of excess unlabeled peptides,indicating that labeled peptides are released from the complexes withoutdissociation of β-2 microglobulin. It should be remembered, however,that the affinity of [¹²⁵I]VSV-8 is about 10-fold lower than that of thenatural peptides. Therefore, this is not necessarily the case for thenatural peptides.

The study using in vitro peptide-binding assay systems suggests thatpeptide binding to Class I molecules is a simple mass action and aligand-receptor interaction. The approach used herein allowscharacterization of the peptide binding specificity to Class I moleculesand of the interaction of peptide-Class I complexes with the T-cellreceptor.

Example 8 Therapeutic Applications

A. Class I Molecule Bank

A reservoir or “bank” of insect cell lines may be established andmaintained, with each cell line expressing one of the 50 to 100 mostcommon Class I MHC heavy chain, a β-microglobulin molecule, as well asat least one assisting molecule. cDNAs encoding these proteins may becloned based on HLA variants obtained from cell lines containingsame—e.g., via the polymerase chain reaction (see Ennis, et al., PNASUSA 87: 2833-7 (1990))—and inserted into the appropriate vector, such asan insect expression vector, to generate cell lines expressing each HLAvariant.

Testing according to the following protocol, for example, can be used todetermine which peptides derived from the virus of choice bind the bestto the different Class I MHC molecules. The various cultures mayappropriately be labeled or catalogued to indicate which Class I MHCmolecules are best for use with particular peptides. Alternatively,transient cultures may be established as needed. As discussed herein,after approximately 48 hours' incubation of a culture of insect cellswith a vector, that culture is apparently capable of expressing emptyMHC molecules which may be loaded with the peptide(s) of choice for thepurpose of activating CD8⁺ cells.

B. Preparation of “Special” Cell Lines

After HLA typing, if insect cell lines expressing the preferred HLA arenot available, cDNAs encoding the preferred HLA and assisting moleculesmay be cloned via use of the polymerase chain reaction. The primersdisclosed in section B.1. above (SEQ ID NO 1 through SEQ ID NO 12) maybe used to amplify the appropriate HLA-A, -B, -C, -E, -F, or -G cDNAs inseparate reactions which may then be cloned and sequenced as describedin the methods disclosed in Example 1, section 1 above. DNA is thenpurified from the PCR reaction using a Gene Clean kit (Bio 101, SanDiego, Calif.) and ligated directly into the Sma I site of pRmHa-3.Individual clones are isolated, the sequences verified, and stableDrosophila cell lines expressing the HLA established. Alternatively, abulk population of recombinant plasmids may be grown in large scale andDNA purified by cesium chloride gradients. The purified DNA is then usedto transfect S2 cells using calcium phosphate precipitation techniques.After 24 hours, the precipitate is washed off the cells and replacedwith fresh Schneider media containing 1 mM CUSO₄. Forty-eight hourslater, the bulk population of transiently transfected cells is used forin vitro activation of CD8⁺ after incubation with syngeneic peptides orprotease digests of specific proteins.

Stable cell lines expressing the cloned HLA may then be established.Alternatively, a population of insect cells transiently expressing abulk population of cloned recombinant molecules from the PCR reactionmay be used for in vitro CD8⁺ activation.

It is also possible to activate haplotype-specific CD8s in vitro usinginsect cells expressing Class I MHC incubated with peptides where thecell line-expressed MHC is not the expressed element in vivo. Thisprovides a unique opportunity to proliferate CD8⁺ cells which recognizea specific antigen associated with a particular MHC which would not bepossible in vivo due to allelic restriction. For example, a peptide (NP)from the nuclear protein of Influenza virus is ordinarily restricted tothe D^(b) molecule; however, we have found that such a peptide can bindto K^(b) (albeit more weakly than to D^(b)) and can generate a degree ofthermal stability to the K^(b) (see FIG. 3). Furthermore,K^(b)-expressing Drosophila cells preincubated with the NP peptide andcocultured with splenocytes from a B6 mouse results in the in vitroactivation of CD8⁺ which specifically recognize the K^(b) moleculeassociated with the NP peptide. In addition, the reciprocal experimentusing a K^(b)-restricted peptide (OVA) derived from ovalbumin andD^(b)-expressing Drosophila cells results in the proliferation of CD8⁺which specifically recognize D^(b) containing the OVA peptide. Such CD8sare able to kill cells (EL4 OVA) transfected with cDNA encoding theovalbumin protein, indicating that in vivo, some D^(b) molecules areloaded with the OVA peptide.

This system therefore provides a unique opportunity to proliferate CD8⁺against specific antigens presented by a Class I molecule which, invivo, is not the restriction element for that peptide. Although enoughantigen is presented in vivo by said Class I for the cell to berecognized by CD8⁺ and killed, it is not enough to proliferate such CD8sin vivo. By loading empty Class I molecules expressed by Drosophilacells with peptide, we are able to override the in vivo restriction byproviding an excess of antigenic peptide to the Class I molecule in anon-competitive environment such that enough antigen is presented by theClass I to activate the specific CD8⁺ recognizing this complex.

C. AIDS Treatment

In vitro activated cells may be administered to patients for in vivotherapy. Preferably, the Class I MHC genotype (haplotype) of theindividual is first determined. Conventional tissue typing isappropriate for this purpose and may be performed at the treatmentcenter or by some appropriate commercial operation. Once theindividual's HLA type(s) is (are) determined, the best combination ofpeptides and Class I MHC molecules suitable for the individual patientis ascertained and prepared as noted above and the appropriate insectcell lines and peptides are provided. Resting or precursor CD8⁺ T-cellsfrom the blood of the patient are then stimulated with the appropriatepeptide-loaded MHC produced by the insect cell culture. Afteractivation, the CD8⁺ cells are reintroduced into the patient'sbloodstream, and the disease process in the patient continues to bemonitored. Methods of removing and re-introducing cellular componentsare known in the art and include procedures such as those exemplified inU.S. Pat. No. 4,844,893 to Honsik, et al. and U.S. Pat. No. 4,690,915 toRosenberg.

Additional treatments may be administered as necessary until the diseaseis sufficiently remediated. Similar treatment protocols are appropriatefor use with other immunosuppressed individuals, including transplantpatients, elderly patients, and the like.

D. Cancer Treatment

In cancer patients, a treatment procedure similar to that describedabove is utilized. However, in such patients, it should be anticipatedthat conventional therapy to reduce the tumor mass may precede theimmune therapy described herein. Therefore, it is preferred that bloodsamples from the putative patient be obtained and stored (e.g. viafreezing) prior to the commencement of conventional therapy such asradiation or chemotherapy, which tends to destroy immune cells. Sincefew, if any, forms of cancer arise in direct response to viralinfection, target peptides for immune treatment are less readilyobserved. However, recent studies indicate that mutations in theoncogenes ras, neu, and p53 contribute to cancer in as much as 50% ofall cancer cases. Thus, peptides derived from these mutated regions ofthe molecules are prime candidates as targets for the present therapy.Pursuant to the protocols disclosed herein, the best combination ofpeptides and Class I molecules for the individual patient may bedetermined and administered.

For example, many tumors express antigens that are recognized in vitroby CD8⁺ cells derived from the affected individual. Such antigens whichare not expressed in normal cells may thus be identified, as well as theHLA type that presents them to the CD8⁺ cells, for precisely targetedimmunotherapy using the methods of the present invention. For example,van der Bruggen, et al. have described an antigen whose expression isdirected by a specific gene and which antigen appears to be presented byHLA A1 (Science 254: 1643-1647 (1991)). As various human tumor antigensare isolated and described, they become good candidates forimmunotherapeutic applications as described herein.

In another, alternative therapeutic mode, it may be feasible toadminister the in vitro activated CD8⁺ cells of the present invention inconjunction with other immunogens. For example, the Large MultivalentImmunogen disclosed in U.S. Pat. No. 5,045,320 may be administered inconjunction with activated CD8⁺ cells.

It is also possible that cytokines such as IL-2 and IL-4, which mediatedifferentiation and activation of T-cells, may be administered as well,as cytokines are able to stimulate the T-cell response against tumorcells in vivo. It is believed that IL-2 plays a major role in the growthand differentiation of CD8⁺ precursors and in CD8⁺ proliferation. Theadministration of IL-2 to cancer patients is frequently associated withan improved anti-tumor response which is likely related to induction oftumor-specific T-cells. However, the best therapeutic effects of IL-2might be obtained by continuous local rather than systemicadministration of IL-2, thus minimizing the IL-2 toxicity and prolongingits biological activity. One may achieve local delivery via transfectingtumor cells with an IL-2 gene construct.

IL-2 cDNA is constructed as described by Karasuyama and Melchers in Eur.J. Immunol. 18: 97-104 (1988). The complete cDNA sequence of IL-2 isobtained as an Xho I fragment from the plasmid pBMGneo IL-2 (seeKarasuyama and Melchers, supra) and directly ligated into the Sal I sitein pRmHa-3. Recombinant pRmHa-3 plasmid with the insert in the correctorientation (determined via restriction mapping with Hind III) ispurified by cesium gradients and used to cotransfect S2 cells using thecalcium phosphate technique. (A mixture of plasmid DNA was prepared forthis purpose: 10 μg pRmHa-3 containing IL-2 cDNA, 6 μg each of pRmHa-3plasmid containing MHC Class I heavy chain or β-2 microglobulin and 2 μgof phshsneo DNA.) Stable cell lines which are inducible via CUSO₄ toexpress heavy chain, β-2 microglobulin and IL-2 were obtained by growingthe transfectants in G418 medium. These stable cell lines were coatedwith peptide and used in the in vitro assay as described above. Tumorcells transfected with IL-2 are observed to enhance the CTL (CD8)activity against the parental tumor cells and bypass CD4 and T helperfunction in the induction of an antitumor or cytotoxic response in vivo.Therefore, increasing the potential of the Drosophila system viacotransfection with the IL-2 gene is suggested herein.

Example 9 Dose Dependence of the Production of Activated T-Cells Usingthe Antigen-Presenting System

Antigen-presenting cells (APC) were produced by transfecting Drosophilacells as described in Example 3 and then tested for their capacity topresent antigen to T-cells from the 2C line of transgenic mice. Withmouse cells as antigen-presenting cells, this line displays strongalloreactivity for L^(d) molecules complexed with an endogenous 8-merpeptide, p2Ca (Leu-Ser-Pro-Phe-Pro-Phe-Asp-Leu, SEQ ID NO 46), derivedfrom a Krebs cycle enzyme, 2-oxoglutarate dehydrogenase (OGDH). The p2Capeptide is exposed naturally bound to L^(d) on the surface of H-2^(d)cells such as B10.D2 cells. The p2Ca peptide has intermediate bindingaffinity for soluble L^(d) molecules (4×10⁶ M⁻¹) and high affinity for2C TCR molecules (2×10⁶ M⁻¹ to 1×10⁷ M⁻¹).

A closely-related 9-mer peptide, QL9(Gln-Leu-Ser-Pro-Phe-Pro-Phe-Asp-Leu, SEQ ID NO 47), has even higheraffinity for these molecules (2×10⁸ M⁻¹ for L^(d) and 2×10⁷ M⁻¹ for 2CTCR). Except for one additional amino acid (glutamine) at the Nterminus, QL9 has an identical sequence to p2Ca and, like p2Ca, formspart of the native sequence of OGDH.

With p2Ca and QL9 peptides (prepared in synthetic form),antigen-presenting cell requirements for mature unprimed 2C CD8⁺ cellswere studied in vitro. The responder CD8⁺ cells were purified frompooled lymph nodes (LN) of 2C mice on a C57BL/6 (B6, H-2^(b)) backgroundby first removing CD4⁺ cells, class II-positive cells and B cells bymAb+complement treatment followed by positive panning.

Cell suspensions were prepared from pooled cervical, axillary, inguinaland mesenteric LN of young adult mice using a tissue grinder. For cellpurification, LN cells were first treated with a cocktail of mAbs(anti-CD4, anti-HSA, anti-I-A^(b)) plus complement for 45 minutes at 37°C. The surviving cells were further separated into CD8⁺ and CD8⁻ (CD4⁻)cells by panning at 4° C. for 60-90 minutes on petri dishes coated withanti-CD8 mAb. The attached CD8⁺ cells were recovered by incubation at37° C. for 5 minutes followed by vigorous pipetting. Non-attached cellswere eluted and treated with anti-CD8 mAb and complement to obtain CD8⁻1B2⁺ 2C cells. More than 95% of the CD8⁺ cells obtained wereclonotype-positive (IB2⁺) and 98% of these cells displayed a naive(CD44^(lo)) phenotype.

TCR stimulation elicited a complex pattern of intracellular events whichlead to early up-regulation of CD69 and CD25 (IL-2 receptor or IL-2R) onthe cell surface. These changes are apparent within a few hours ofstimulation and are followed by cytokine synthesis and cellproliferation. Drosophila cells were transfected with genes for L^(d),L^(d) and B7.1 (L^(d).B7), L^(d) and ICAM-1 (L^(d).ICAM) or L^(d), B7.1and ICAM-1 (L^(d).B7.1CAM). FIG. 11 shows CD69 and CD25 expression onpurified naive CD8⁺ 2C cells stimulated for 12 hours with transfectedDrosophila cells and p2Ca versus QL9 peptide at a concentration of 10μM. Purified CD8⁺ 2C cells were incubated with various Drosophila cellsplus a peptide (either p2Ca or QL9, 10 μM) in bulk (2 ml) culture for 12hours and then stained for CD69 or CD25. Either p2Ca or QL9 presented byDrosophila cells transfected with L^(d).B7, L^(d).ICAM or L^(d).B7.ICAMwere effective in stimulating the up-regulation of CD69 and CD25.However, non cultured 2C cells without either peptide or Drosophilacells did not show up-regulation of CD69 and CD25 (top panel).

In the presence of Drosophila cells expressing L^(d) alone, induction ofCD69 and CD25 expression on CD8⁺ 2C cells was low, but significant, withthe strong QL9 peptide but barely detectable with the weaker p2Capeptide. With L^(d).B7 or L^(d).ICAM antigen-presenting cells, bothpeptides elicited marked up-regulation of CD69 and CD25. However,L^(d).B7.ICAM antigen-presenting cells induced even higher expression ofthese molecules.

Drosophila cells transfected with L^(d) alone failed to causeproliferation of 2C CD8⁺ cells to either p2Ca or QL9 peptide in theabsence of exogenous lymphokines, consistent with the minimal capacityof these cells as antigen-presenting cells to induce CD69 and CD25expression. The results are shown in FIG. 12. Responses to p2Ca (above)and QL9 (below) were measured by culturing 5×10⁴ purified CD8⁺ 2C cellswith 2×10⁵ Drosophila cells in the presence or absence of the indicatedconcentrations of peptides for 3 days. [³H] TdR was added during thelast 8 hours of culture; rIL-2 was added at a final concentration of 20units/ml. The data are the mean of triplicate cultures. Whensupplemented with exogenous IL-2, however, both peptides stimulatedsignificant proliferative responses at high doses (10 μM). Theproliferative responses elicited by QL9 were far stronger than for p2Ca(note the large difference between the scales of x-axes of the upper andthe lower panel).

Dose-response relationships for proliferation on day 3 elicited byDrosophila cells transfected with L^(d).B7.1CAM and presenting p2Capeptide were compared to those elicited by Drosophila cells transfectedwith L^(d).B7, L^(d).ICAM or L^(d).B7.ICAM presenting QL9 peptide. Theresults (means of triplicate cultures) are shown in FIG. 13. DrosophilaAPC (2×10⁵ cells) were cultured with 5×10⁴ CD8⁺ 2C cells for three daysin the presence or absence of peptides. [³H] TdR was added during thelast 8 hours of culture; no IL-2 was added to the cultures.

Optimal proliferative responses elicited by L^(d).B7.ICAMantigen-presenting cells on day 3 required high concentrations of p2Capeptide, e.g. 10 μM (10⁻⁵ M), (FIG. 13). The results for QL9 peptidewere different. The does-response curves for L^(d).B7 antigen-presentingcells plus QL9 and L^(d).ICAM antigen-presenting cells plus QL9approximated the results for L^(d).B7.ICAM antigen-presenting cells plusp2Ca (FIG. 13). However, with L^(d).B7.ICAM antigen-presenting cells,proliferative responses to QL9 on day 3 were maximal with 100 nM (10⁻⁷M) and were clearly apparent with doses as low as 10 pM (10⁻¹¹ M) (FIG.13). At high doses, e.g. 10 μM (10⁻⁵ M), QL9 inhibited the proliferativeresponse on day 3 (FIG. 13, note the log scale).

Inhibition of proliferation by L^(d).B7.ICAM antigen-presenting cellsand QL9 peptide did not apply to IL-2 production (FIG. 14, right), andwas only seen with high doses of antigen-presenting cells (FIG. 14,left). L^(d).B7.ICAM antigen-presenting cells plus QL9 peptide was againfound to be more effective in eliciting IL-2 production thanL^(d).B7.ICAM antigen-presenting cells plus p2Ca peptide (FIG. 14,right). The data indicates that antigen-presenting cells withoutpeptides were ineffective in eliciting either response over the 100-foldrange of antigen-presenting cells density tested (FIG. 14, “-pep,” opensquares).

Changes in the dose-response relationships of the responses of CD8⁺ 2Ccells on Day 3, Day 4 and Day 5 were examined using QL9 peptide withL^(d).B7, L^(d).ICAM or L^(d).B7.ICAM antigen-presenting cells. Theresults are shown in FIG. 15. Responses were measured with 5×10⁴ CD8⁺ 2Ccells and 3×10⁵ antigen-presenting cells at the indicated peptideconcentrations. The data are the mean results of triplicate cultures.

With an intermediate dose of 100 nM (10⁻⁷ M) QL9 peptide, proliferativeresponses to L^(d).B7.1CAM antigen-presenting cells were high on Day 3(FIG. 15, left), reached a peak on Day 4 (FIG. 15, center) and thendeclined to low levels on Day 5 (FIG. 15, right). With a high dose of 10μM (10⁻⁵ M) QL9 peptide, however, the response was low on Day 3 (FIG.15, left), but then increased markedly to reach a high peak on Day 5(FIG. 15, right).

Transient inhibition of proliferation induced by QL9 on Day 3 was onlyseen when the avidity of T-cell/APC interaction was very high.Decreasing the avidity of T-cell/APC interaction by using lower doses ofAPC (FIG. 14, left) or lower doses of QL9 peptide (FIG. 15, left)augmented the Day 3 proliferative response. Reducing the avidity ofT-cell/APC interaction enhanced the early (Day 3) proliferative responsebut reduced the late (Day 5) response and also reduced IL-2 production(FIG. 16, right). In FIG. 16 the results obtained using CD8⁺ 2C and CD8⁻2C cells on Days 2, 3, 4 and 5 are compared.

The observations apply with the highly immunogenic L^(d).B7.ICAMantigen-presenting cells. However, with the less immunogenic L^(d).B7 orL^(d).ICAM antigen-presenting cells, proliferative responses to QL9peptide required high doses of peptide (FIG. 13, FIG. 15) and werecrucially dependent upon CD8 expression by the responder cells (FIG.16). These responses with L^(d).B7 and L^(d).ICAM antigen-presentingcells reached a peak on Days 3 or 4 (rather than Day 5) and were farlower than with L^(d).B7.ICAM antigen-presenting cells. With low dosesof QL9, e.g. 1 nM (10⁻⁹ M), proliferative responses with L^(d).B7 andL^(d).ICAM antigen-presenting cells were completely undetectable (<100cpm) (FIG. 13). This was in striking contrast to the results seen usingL^(d).B7.ICAM antigen-presenting cells, where 1 nM QL9 led to highresponses (>10,000 cpm) (FIG. 13). In contrast to the results with highdoses of QL9 peptide (10 μM, Table 2), the synergistic interactionbetween B7 and ICAM for proliferative responses became pronounced at lowpeptide doses.

As can be seen, L^(d).B7.ICAM cells act as extremely potentantigen-presenting cells for naive 2C cells. Raising the avidity ofT-cell/APC interaction to a high level inhibits the early proliferativeresponse but potentiates the late response and IL-2 production isenhanced. For the high-affinity peptides such as QL9, the synergybetween B7 and ICAM is pronounced at low doses of antigen.

Example 10 Production of Cytotoxic T-Cells Using the Antigen PresentingSystem

Antigen-presenting cells (APC) were produced by transfecting Drosophilacells as described in Example 3 and then tested for their ability toinduce CTL activity. CTL activity was tested on ⁵¹Cr-labeled RMA.S-L^(d)targets sensitized with QL9 peptide, no peptide or an irrelevant peptide(MCMV). CD8⁺ 2C cells (5×10⁵) were cultured with 2×10⁶ transfectedDrosophila cells in a volume of 2 ml in a 24-well culture plate. Cai,Z., and J. Sprent, (1994). Peptides were present during the culture at aconcentration of 10 mM. After 4 days, the cells were pooled and adjustedto the required number. To prepare targets, RMA-S.L^(d) cells werelabeled with ⁵¹Cr (100 mCi/1-2×10⁶ cells) at 37° C. for 90 minutes inthe presence or absence of peptides. After labeling, the cells werethoroughly washed and resuspended in medium with or without peptides.Specific ⁵¹Cr release was calculated according to established procedure.Id.

The capacity of L^(d).B7, L^(d).B7.ICAM and L^(d).ICAMantigen-presenting cells to induce CTL activity to 10 μM QL9 peptide inbulk cultures is shown in FIG. 17. Strongly immunogenic L_(d).B7.ICAMantigen-presenting cells were efficient in generating QL9-specific CTL(FIG. 17, center). Significantly, L^(d).B7 antigen-presenting cells werealso effective in generating CTL to QL9 (FIG. 17, left). The surprisingfinding, however, was that L^(d).ICAM antigen-presenting cells weretotally unable to stimulate CTL generation (FIG. 17, right). This result(representative of three different experiments) was unexpected, becauseL^(d).ICAM antigen-presenting cells were no less efficient than L^(d).B7antigen-presenting cells at inducing proliferative responses to QL9(FIGS. 13 and 15). The L^(d).ICAM-stimulated cultures in FIG. 17contained large numbers of blast cells, and total cell yields were about3-fold higher than the input number.

The surprising finding that L^(d).ICAM antigen-presenting cells weretotally unable to stimulate CTL generation applied to cultures notsupplemented with exogenous lymphokines. However, when exogenous IL-2was added to the cultures, L^(d).ICAM antigen-presenting cells inducedstrong CTL activity to QL9 (FIG. 18, right; 20 u/ml exogenous IL-2).

Example 11 Proliferation of Normal T-Cells Induced by theAntigen-Presenting System

Antigen-presenting cells (APC) were produced by transfecting Drosophilacells as described in Example 3 and then tested for their ability toinduce proliferation in normal (nontransgenic) murine T-cells.

The capacity of L^(d).B7.ICAM Drosophila cells to induce a strongprimary response of 2C TCR transgenic CD8⁺ cells raised the questionwhether Drosophila cells could act as antigen-presenting cells fornormal (nontransgenic) CD8⁺ cells. Since the 2C mice were on a B6 (H-2b)background, the response of normal B6 CD8⁺ cells was tested. Gradeddoses of CD8⁺ cells from normal B6 mice were cultured with 10 μM QL9peptide presented by L^(d).B7.ICAM Drosophila antigen-presenting cells,i.e. a situation where a diverse repertoire of T-cells was exposed toonly a single alloantigen (L^(d)+QL9) but at high concentration. Asshown in FIG. 19 (left), presentation of QL9 by L^(d).B7.ICAMantigen-presenting cells was indeed immunogenic for normal B6 CD8⁺ cellsand led to appreciable proliferative responses on Day 3 (80,000 cpm)with large doses of responder cells (1×10⁶) in the absence of addedcytokines; responses to an unrelated peptide, MCMV, were much lower(though significant) and no response occurred in the absence of peptide.As expected, the response of normal B6 CD8+ cells to QL9 plusL^(d).B7.ICAM antigen-presenting cells was substantially lower than withnormal B10.D2 spleen cells as antigen-presenting cells (where theallostimulus was provided by a multiplicity of self peptides bound toL^(d), K^(d) and D^(d), but at low concentration) (FIG. 19, right. Notethe difference in the Y axis scales).

Example 12 Activation of Cytotoxic T-Cells Using Immobilized PurifiedRecombinant MHC Class I Molecules and Assisting Molecules

Except as noted, cells, materials and reagents were prepared asdescribed in Example 4. Biotinylated anti-mouse CD28 monoclonal antibody(clone 37.51) was purchased from Pharmingen (San Diego, Calif.). Thisantibody is also available from Caltag (South San Francisco, Calif.).This antibody, which binds to CD28 co-stimulatory receptor, a ligand ofB7.1 and B7.2 on the surface of T-cells, augments the proliferation ofT-cells (Gross et al. J. Immunol. 149, 380-388, 1992). IL-2 was used asa concanavalin A supernatant (10% final concentration).

Substrates were prepared as follows: Fifty microliters of PBS containing1 microgram/ml of neutravidin were added to each well of a 96 well cellculture plate (Corning cat.#25860). After 2 hours at room temperature,the wells were washed 3 times with PBS prior to incubation withbiotinylated molecules. Avidin-coated mouse red blood cells wereprepared as described for avidin-coated sheep red blood cells in Example4. Avidin-coated latex beads were prepared as described Example 4.

Biotinylation of recombinant L^(d) was performed as described in Example4. Biotinylated recombinant L^(d) was immobilized on the substratetogether with biotinylated anti-CD28 antibody. Avidin-coated red bloodcells or latex beads were incubated with 0.2 mg/ml biotinylated L^(d),0.025 mg/ml biotinylated anti-CD28, or a mixture thereof for 30 minutesat room temperature, then washed 3 times in DMEM containing 10% FCS andused immediately. Avidin-coated 96 well plates were incubated with 50microliters per well of 2 microgram/ml biotinylated L^(d), 0.25microgram/ml anti-CD28, or mixtures thereof for 30 minutes at roomtemperature, then washed 3 times using DMEM containing 10% FCS and usedimmediately.

2C+ T-cells were prepared as described in Example 4. CD8⁺ cells fromC57BL/6 mice were prepared from the lymph nodes of these mice bymagnetic depletion. Purified cells were consistently 90-92% positive forCD8 expression, as assessed by flow cytofluorometry.

T-cell activation was performed in culture plates coated with purifiedL^(d) molecules and anti-CD28 antibody. T-cells and peptide (0.02 mMfinal concentration) were added to 96 well plates coated with L^(d)and/or anti-CD28, in a final volume of 0.2 ml/well and cultured for theappropriate time at 37° C. in humid atmosphere containing 5% Co₂.

T-cell activation using red blood cells or latex beads coated withpurified L^(d) molecules and anti-CD28 antibody was performed inuncoated culture plates. T-cells and peptide (0.02 mM) were added toeach well, together with 100,000 red blood cells or latex beads. Finalvolume was 0.2 ml. Culture conditions were as described above.

T-cell mitogenesis was assayed by incorporation of a pulse of 1 pCi oftritiated thymidine per well by triplicate cell cultures. Cells wereharvested 8 hours later and thymidine incorporation was determined bycounting the filters in a scintillation counter.

RMA.S cells (target cells) expressing L^(d) were incubated withradiolabeled chromium for 90 minutes at 37° C., washed 3 times anddistributed in 96 well U-bottom plate (Costar cat#3799) (5000-10000cells per well) in the presence of the appropriate peptide at 0.01 mM.Various amounts of activated T-cells (effector cells) were added toreach effector/target ratios (E/T ratios) ranging between 150 and 1.After 5 hours of incubation at 37° C., 0.1 ml of supernatant wascollected from each well and counted in a gamma counter. Percent lysiswas determined by a standard method (Coligan et al., Current Protocolsin Immunology, section 3.11, Wiley, New York (1991)).

Immobilized purified L^(d) and anti-CD28 antibody were mitogenic for 2C+T-cells. When QL9 peptide was used, a thymidine incorporation above100,000 cpm per well was consistently measured by day 3-5 of culture(FIG. 20). Maximum thymidine incorporation in that same range wasobtained using any of the three methods of activation (moleculesimmobilized on plastic, on red blood cells or on latex beads). Whenactivation molecules were immobilized on plastic, L^(d) aloneimmobilized on plastic induced a transient mitogenesis (FIG. 20, brokenline) whereas L^(d) plus anti-CD28 antibody induced a higher and moresustained mitogenesis (FIG. 20, solid line). Anti-CD28 antibody inaddition to L^(d) was required for the induction of any mitogenesis inthe model using red blood cells. However, anti-CD28 antibody alone didnot induce any mitogenesis.

QL9-L^(d) complexes are recognized by the 2C T-cell receptor with a highaffinity. Complexes of other peptides and L^(d) recognized by thisreceptor with a lower affinity were able to activate 2C+ T-cells; theseincluded peptides p2Ca and SL9 (FIG. 21). However, IL-2 added at day 2of culture was required in order to observe mitogenesis using thesepeptides. Activation was peptide specific since LCMV peptide, a controlpeptide that is not recognized by the 2C T-cell receptor, did not induceactivation. Mitogenesis was measurable starting with as little as 700T-cells per well (96 plate well), demonstrating that the methodactivated a very small number of T-cells in a peptide specific manner.

2C+ T-cells were mixed with total CD8⁺ cells from C57BL/6 mice at a 1/99ratio and cells were cultured with immobilized L^(d) and anti-CD28antibody in the presence of peptide QL9. IL-2 was added at day 2 ofculture, and subsequently every other day. Cell activation andproliferation was noted, as evidenced by the formation of clumps,presence of enlarged cells that progressively spread in the well. At day12 of culture, cells were harvested and percentage of cells expressingthe 2C T-cell receptor was assessed by staining with the anticlonotypicantibody 1B2 and analysis by flow cytofluorometry: 43% of the cellscultured initially in the presence of peptide QL9 were 2C+, (FIG. 22)whereas 49% of the cells expressed 2C after stimulation with peptidep2Ca (FIG. 23), and 22% with peptide SL9 (FIG. 24). This shows aconsiderable enrichment (43, 49 and 22 times) in specific T-cells after12 days of culture. Enrichment was observed even with SL9, a peptidethat makes a low affinity complex with the 2C T-cell receptor. Thismethod thus can specifically activate and enrich a small subpopulationof antigen specific T-cells out of a heterogenous mixture of T-cells.

2C+ T-cells, activated using L^(d) and anti-CD28 antibody in thepresence of QL9 peptide, were cultured for 5 days, then their cytotoxiccapacity was assessed. The results, shown in FIG. 25, demonstrated thatimmobilized activation molecules induced the cells to differentiate intoeffector cytotoxic T-cells. Lysis was specific, since it was observed inthe presence of QL9 peptide but not in the presence of a control peptide(LCMV). Resting T-cells were thus activated using immobilized moleculesto differentiate into cytotoxic T-cells able to specifically killtargets.

As can be seen, immobilized purified MHC Class I and assisting moleculescan be used to specifically activate naive resting T-cells intocytotoxic T-cells. MHC class I and assisting molecules are sufficientfor activation; no additional signal originating from antigen-presentingcells is necessary. MHC class I and assisting molecules immobilized onsubstrates, such as cell culture plates, provide an appropriate tool forT-cell activation. Several advantages are offered by such coatedsubstrates. They are easy to prepare and to manipulate, they can becoated with well-controlled amounts of molecules, ensuring reproducibleactivation conditions.

Example 13 Activation of Human Cytotoxic T-Cells

A unit of human blood (450 mls) collected in heparin (10 μ/ml) wasobtained through the General Clinical Research Center (GCRC) at ScrippsClinic, La Jolla, Calif. The blood was first processed in aFicoll-Hypaque density gradient in a 50 cc conical centrifuge(Histopaque 1077, Sigma) according to the manufacturer's specifications.Once the buffy coat was obtained, the sample was washed in buffer 1(D-PBS without Ca⁺⁺ or Mg⁺⁺), then buffer 2 (RPMI with 4% fetal calfserum (FCS)) and a final wash in buffer 3 (D-PBS+1% human serum albumin(HSA, 25% Buminate/Baxter-Hyland) and 0.2% sodium citrate (w/v)).

The total peripheral blood mononuclear cell (PBMCs) preparation wascounted from the washed cells. This preparation was then taken through aMAXSEP isolation procedure (Baxter) where CD8⁺ cells were selected bynegative selection. A cocktail of mabs to cells targeted for removal(CD19-PharMingen, CD4-Ortho-mune, CD15-PharMingen, CD56-PharMingen,CD14-PRI) was prepared at 2 μg/ml of cells. The total PBMC cell countwas diluted to 20×10⁶/ml in buffer 3 and the antibodies were added.

The mixture (approximately 40 mls) was rotated (4 rpm) on a rotaryshaker at 4° C., so that the tube mixed the sample end over end, for atotal of 30 minutes. After the sensitization phase, the cells werewashed with buffer 3 and resuspended in the same buffer with magneticDynal beads coated with sheep-anti-mouse IgG (SAM) (Dynabeads M450#110.02). The stock of beads is usually 4×10⁸ beads/ml. The finalbead:target cell ratio is 10:1.

To determine the final volume of beads to use, the formulas below werefollowed:

(Total cells sensitized)×(% of population of target cells)=Totaltheoretical target cell number.

 Total theoretical target cell number×10=Total beads required.

Total beads required/Bead concentration of stock=Volume of beadsrequired.

The volume of the final sensitized cells:beads mixture was approximately50 mls. The mixture was put into a 150 ml Fenwal Transfer Pack(#4R2001), air added with a needle and the mixture rotating undersimilar conditions as above (30 minutes, 4° C., 4 rpm, agitatedend-over-end). At the end of the incubation period, the target cellswere removed with a MAXSEP separation device according to themanufacture's specifications. The separated cells were transferred fromthe bag and counted to determine recovery. FACS analysis was performedto determine the purity of the sample.

The resulting separated human CD8⁺ cells were stimulated with Drosophila(fly) antigen-presenting cells which were transfected and shown toexpress human HLA A2.1, B7.1 and/or LFA-3. The fly cells were diluted to10⁶/ml in Schneider's medium with 10% FCS serum. On the following dayCuSO4 was added to the cultured cells which were incubated for 24 hoursat 27° C. and harvested. The harvested cells were washed and suspendedin Insect X-press medium with a final peptide concentration of 100 μg/mland incubated for 3 hours at 27° C.

The peptides used were HIV-RT (ILKEPVHGV) (SEQ ID NO 48), Tyrosinase(YMNGTMSQV) (SEQ ID NO 49), and Influenza Matrix (GILGFVFTL) (SEQ ID NO50).

The control peptide was derived from the core of the hepatitis virus andhad the sequence FLPSDFFPSV (SEQ ID NO 51).

The fly antigen-presenting cells were added to the CD8⁺ cells at a ratioof 1:10 (APC:CD8⁺ T-cell). The cells were incubated in flat bottom wells(48 wells) for four days at 37° C. At day 5, IL-2 (10 μ/ml) and IL-7 (10μ/ml) (Genzyme) were added with a media change. On day 11 a CTL assaywas performed.

JY, an EBV-transformed human B cell line expressing HLA 2.1, B7 was usedas the target cell in the chromium release assay. Vissereu, M. J. W. etal. J. Immunol., 154:3991-3998 (1995). JY cells were seeded at 3×10⁵cells/ml in RPMI with 10% FCS, 24 hours prior to the assay. The JY cellswere counted and washed once in RPMI wash solution (4% Rehautin FCS, 1%HEPES, 0.025% gentamycin in RPMI) to provide 5×10 cells per sample. Thecell pellet was gently resuspended in 100 μl of ⁵¹Chromium stock (0.1mCi, NEN) and placed in a 37° C. water bath for 1 hour with agitationevery fifteen minutes. Labeled JY cells were washed four times for 6minutes each at 1400 rpm in 10 mls of RPMI wash solution. Cells wereadjusted to 1×10 ⁵ cells/ml in RPMI/10% Hyclone. Efficiency of targetcell labelling is confirmed by standard gamma counter techniques.

For peptide loading of target cells, two mls of labeled JY cells (2×10⁵cells) were incubated with 10 μg/ml peptide for 30 minutes at roomtemperature. Peptide stock solutions (1 mg/ml) were stored at −70° C. Ina 96 well round bottom plate, 100 μl effector cells and 100 μl peptideloaded target cells were combined at ratios of 84:1, 17:1 and 3.4:1(effector:target). Controls to measure spontaneous release and maximumrelease of ⁵¹Cr were included in duplicate. Samples were incubated at37° C. for 6 hours.

K562 cells at a concentration of 10⁷/ml in RPMI with 10% FCS were addedat a ratio of 20:1 (unlabeled K562:labeled JY). This erythroleukemiccell line was used to reduce NK background cell lysis in the chromiumrelease assay. Plates were centrifuged at 1000 rpm for 5 minutes and 100μl supernatant from each sample transferred to 96 tube collection tubes.Analysis of cell lysis is determined by standard gamma countingtechniques (Gammacell 1000, Nordion).

CTL activity produced by activating human CD8⁺ T-cells withantigen-presenting cells loaded with influenza matrix peptide (SEQ ID NO50) is shown in FIG. 26. Prior exposure to influenza virus indicatesthat this CTL activity was a secondary response. Data points are themean of values from triplicate cultures. Antigen-presenting cellsexpressing A2.1, B7.1 and ICAM-1 were more effective thanantigen-presenting cells expressing A2.1 and B7.1 or antigen-presentingcells expressing A2.1, B7.1 and LFA-3.

The results of activating human CD8⁺ T-cells with antigen-presentingcells loaded with HIV-RT peptide (SEQ ID NO 48) are presented in FIG.27. Since screening of the blood of this patient had indicated no priorexposure to HIV, these results indicate the CTL activity was based on aprimary response. In this case, only antigen-presenting cells expressingA2.1, B7.1 and ICAM-1 produced CTL activity that was significantlygreater than control levels.

FIG. 28 shows the CTL activity of human CD8⁺ T-cells activated byantigen-presenting cells loaded with tyrosinase peptide (SEQ ID NO 49).Tyrosinase is a normally occurring enzyme that is over-expressed inmelanoma tumor cells. Again, the antigen-presenting cells expressing allthree molecules, ICAM-1 as well as A2.1 and B7.1, were most effective,especially at the lowest effector:target ratio tested.

These results show that the antigen presenting system is capable ofproducing effective CTL activity in human CD8⁺ T-cells directed againsta peptide that is derived from an endogenous protein that isover-expressed in tumor cells. This also demonstrates that this in vitrostimulation of CD8⁺ T-cells using both B7 and ICAM will generatecytotoxic CD8⁺ T-cells even against peptides that would otherwise berecognized as self. This is contrary to present knowledge that such“self” peptides could not be used to create cytotoxic T-cells. Thismethod greatly expands the number of possible tumor specific antigensthat can be used to activate CD8⁺ T-cells against the tumor.

The foregoing is intended to be illustrative of the present invention,but not limiting. Numerous variations and modifications may be effectedwithout departing from the true spirit and scope of the invention.

65 1 23 DNA Artificial Sequence Synthetic PCR primer (SPP) 1 ccaccatggccgtcatggcg ccc 23 2 23 DNA Artificial Sequence Synthetic PCR primer(SPP) 2 ggtcacactt tacaagctct gag 23 3 23 DNA Artificial SequenceSynthetic PCR primer (SPP) 3 ccaccatgct ggtcatggcg ccc 23 4 23 DNAArtificial Sequence Synthetic PCR Primer (SPP) 4 ggactcgatg tgagagacacatc 23 5 23 DNA Artificial Sequence Synthetic PCR Primer (SPP) 5ccaccatgcg ggtcatggcg ccc 23 6 23 DNA Artificial Sequence Synthetic PCRPrimer (SPP) 6 ggtcaggctt tacaagcgat gag 23 7 23 DNA Artificial SequenceSynthetic PCR Primer (SPP) 7 ccaccatgcg ggtagatgcc ctc 23 8 23 DNAArtificial Sequence Synthetic PCR Primer (SPP) 8 ggttacaagc tgtgagactcaga 23 9 23 DNA Artificial Sequence Synthetic PCR Primer (SPP) 9ccaccatggc gccccgaagc ctc 23 10 23 DNA Artificial Sequence Synthetic PCRPrimer (SPP) 10 ggtcacactt tattagctgt gag 23 11 23 DNA ArtificialSequence Synthetic PCR Primer (SPP) 11 ccaccatggc gccccgaacc ctc 23 1223 DNA Artificial Sequence Synthetic PCR Primer (SPP) 12 ggtcacaatttacaagccga gag 23 13 427 DNA Drosophila Melanogaster 13 aattcgttgcaggacaggat gtggtgcccg atgtgactag ctctttgctg caggccgtcc 60 tatcctctggttccgataag agacccagaa ctccggcccc ccaccgccca ccgccacccc 120 catacatatgtggtacgcaa gtaagagtgc ctgcgcatgc cccatgtgcc ccaccaagag 180 ttttgcatcccatacaagtc cccaaagtgg agaaccgaac caattcttcg cgggcagaac 240 aaaagcttctgcacacgtct ccactcgaat ttggagccgg ccggcgtgtg caaaagaggt 300 gaatcgaacgaaagacccgt gtgtaaagcc gcgtttccaa aatgtataaa accgagagca 360 tctggccaatgtgcatcagt tgtggtcagc agcaaaatca agtgaatcat ctcagtgcaa 420 ctaaagg 42714 740 DNA Drosophila Melanogaster 14 attcgatgca cactcacatt cttctcctaatacgataata aaactttcca tgaaaaatat 60 ggaaaaatat atgaaaattg agaaatccaaaaaactgata aacgctctac ttaattaaaa 120 tagataaatg ggagcggctg gaatggcggagcatgaccaa gttcctccgc caatcagtcg 180 taaaacagaa gtcgtggaaa gcggatagaaagaatgttcg atttgacggg caagcatgtc 240 tgctatgtgg cggattgcgg aggaattgcactggagacca gcaaggttct catgaccaag 300 aatatagcgg tgtgagtgag cgggaagctcggtttctgtc cagatcgaac tcaaaactag 360 tccagccagt cgctgtcgaa actaattaagttaatgagtt tttcatgtta gtttcgcgct 420 gagcaacaat taagtttatg tttcagttcggcttagattt cgctgaagga cttgccactt 480 tcaatcaata ctttagaaca aaatcaaaactcattctaat agcttggtgt tcatcttttt 540 ttttaatgat aagcattttg tcgtttatactttttatatt tcgatattaa accacctatg 600 aagttcattt taatcgccag ataagcaatatattgtgtaa atatttgtat tctttatcag 660 gaaattcagg gagacgggga agttactatctactaaaagc caaacaattt cttacagttt 720 tactctctct actctagagt 740 15 60 DNAArtificial Sequence Synthetic PCR Primer (SPP) 15 gcttggatcc agatctaccatgtctcgctc cgtggcctta gctgtgctcg cgctactctc 60 16 36 DNA ArtificialSequence Synthetic PCR Primer (SPP) 16 ggatccggat ggttacatgt cgcgatcccacttaac 36 17 19 DNA Artificial Sequence Synthetic PCR Primer (SPP) 17ggagccgtga ctgactgag 19 18 24 DNA Artificial Sequence Synthetic PCRprimer (SPP) 18 ccctcggcac tgactgactc ctag 24 19 38 DNA ArtificialSequence Synthetic Expression Vector Fragment 19 gatccttatt agatctcaccatcaccatca ccattgag 38 20 38 DNA Artificial Sequence Syntheticexpression vector fragment 20 tcgactcaat ggtgatggtg atggtgagat ctaataag38 21 3875 DNA Artificial Sequence Synthetic Expression Vector Fragment21 ttgcaggaca ggatgtggtg cccgatgtga ctagctcttt gctgcaggcc gtcctatcct 60ctggttccga taagagaccc agaactccgg ccccccaccg cccaccgcca cccccataca 120tatgtggtac gcaagtaaga gtgcctgcgc atgccccatg tgccccacca agagctttgc 180atcccataca agtccccaaa gtggagaacc gaaccaattc ttcgcgggca gaacaaaagc 240ttctgcacac gtctccactc gaatttggag ccggccggcg tgtgcaaaag aggtgaatcg 300aacgaaagac ccgtgtgtaa agccgcgttt ccaaaatgta taaaaccgag agcatctggc 360caatgtgcat cagttgtggt cagcagcaaa atcaagtgaa tcatctcagt gcaactaaag 420gggaattcga gctcggtacc cggggatcct tattagatct caccatcacc atcaccattg 480agtcgacctg caggcatgca agctattcga tgcacactca cattcttctc ctaatacgat 540aataaaactt tccatgaaaa atatggaaaa atatatgaaa attgagaaat ccaaaaaact 600gataaacgct ctacttaatt aaaatagata aatgggagcg gcaggaatgg cggagcatgg 660ccaagttcct ccgccaatca gtcgtaaaac agaagtcgtg gaaagcggat agaaagaatg 720ttcgatttga cgggcaagca tgtctgctat gtggcggatt gcggaggaat tgcactggag 780accagcaagg ttctcatgac caagaatata gcggtgagtg agcgggaagc tcggtttctg 840tccagatcga actcaaaact agtccagcca gtcgctgtcg aaactaatta agttaatgag 900tttttcatgt tagtttcgcg ctgagcaaca attaagttta tgtttcagtt cggcttagat 960ttcgctgaag gacttgccac tttcaatcaa tactttagaa caaaatcaaa actcattcta 1020atagcttggt gttcatcttt ttttttaatg ataagcattt tgtcgtttat actttttata 1080tttcgatatt aaaccaccta tgaagtctat tttaatcgcc agataagcaa tatattgtgt 1140aaatatttgt attctttatc aggaaattca gggagacggg aagttactat ctactaaaag 1200ccaaacaatt tcttacagtt ttactctctc tactctagag tagcttggca ctggccgtcg 1260ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac 1320atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac 1380agttgcgcag cctgaatggc gaatggcgcc tgatgcggta ttttctcctt acgcatctgt 1440gcggtatttc acaccgcata tggtgcactc tcagtacaat ctgctctgat gccgcatagt 1500taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc 1560cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt 1620caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg 1680ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc 1740gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac 1800aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt 1860tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag 1920aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg 1980aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa 2040tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc 2100aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag 2160tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa 2220ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc 2280taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg 2340agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa 2400caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa 2460tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg 2520gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag 2580cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg 2640caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt 2700ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt 2760aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac 2820gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag 2880atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg 2940tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca 3000gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga 3060actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca 3120gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc 3180agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca 3240ccgaactgag atacctacag cgtgagcatt gagaaagcgc cacgcttccc gaagggagaa 3300aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc 3360cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc 3420gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg 3480cctttttacg gtcctggcct tttgctggcc ttttgctcac atgtctttcc tgcgttatcc 3540cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc 3600cgaaccgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa 3660accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga 3720ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc attaggcacc 3780ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca 3840atttcacaca ggaaacagct atgacatgat taccg 3875 22 71 DNA ArtificialSequence Synthetic expression vector fragment 22 gatccttatt agatcttacccatacgacgt cccagattac gctcgatctc accatcacca 60 tcaccattga g 71 23 71 DNAArtificial Sequence Synthetic expression vector fragment 23 tcgactcaatggtgatggtg atggtgagat cgagcgtaat ctgggacgtc gtatgggtaa 60 gatctaataa g71 24 3908 DNA Artificial Sequence Synthetic expression vector fragment24 ttgcaggaca ggatgtggtg cccgatgtga ctagctcttt gctgcaggcc gtcctatcct 60ctggttccga taagagaccc agaactccgg ccccccaccg cccaccgcca cccccataca 120tatgtggtac gcaagtaaga gtgcctgcgc atgccccatg tgccccacca agagctttgc 180atcccataca agtccccaaa gtggagaacc gaaccaattc ttcgcgggca gaacaaaagc 240ttctgcacac gtctccactc gaatttggag ccggccggcg tgtgcaaaag aggtgaatcg 300aacgaaagac ccgtgtgtaa agccgcgttt ccaaaatgta taaaaccgag agcatctggc 360caatgtgcat cagttgtggt cagcagcaaa atcaagtgaa tcatctcagt gcaactaaag 420gggaattcga gctcggtacc cggggatcct tattagatct tacccatacg acgtcccaga 480ttacgctcga tctcaccatc accatcacca ttgagtcgac ctgcaggcat gcaagctatt 540cgatgcacac tcacattctt ctcctaatac gataataaaa ctttccatga aaaatatgga 600aaaatatatg aaaattgaga aatccaaaaa actgataaac gctctactta attaaaatag 660ataaatggga gcggcaggaa tggcggagca tggccaagtt cctccgccaa tcagtcgtaa 720aacagaagtc gtggaaagcg gatagaaaga atgttcgatt tgacgggcaa gcatgtctgc 780tatgtggcgg attgcggagg aattgcactg gagaccagca aggttctcat gaccaagaat 840atagcggtga gtgagcggga agctcggttt ctgtccagat cgaactcaaa actagtccag 900ccagtcgctg tcgaaactaa ttaagttaat gagtttttca tgttagtttc gcgctgagca 960acaattaagt ttatgtttca gttcggctta gatttcgctg aaggacttgc cactttcaat 1020caatacttta gaacaaaatc aaaactcatt ctaatagctt ggtgttcatc ttttttttta 1080atgataagca ttttgtcgtt tatacttttt atatttcgat attaaaccac ctatgaagtc 1140tattttaatc gccagataag caatatattg tgtaaatatt tgtattcttt atcaggaaat 1200tcagggagac gggaagttac tatctactaa aagccaaaca atttcttaca gttttactct 1260ctctactcta gagtagcttg gcactggccg tcgttttaca acgtcgtgac tgggaaaacc 1320ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata 1380gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatggc 1440gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atatggtgca 1500ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac 1560ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga 1620ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac 1680gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt 1740agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct 1800aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat 1860attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg 1920cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg 1980aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc 2040ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat 2100gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact 2160attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca 2220tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact 2280tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg 2340atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg 2400agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg 2460aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg 2520caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag 2580ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc 2640gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga 2700tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat 2760atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc 2820tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag 2880accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct 2940gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac 3000caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc 3060tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg 3120ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt 3180tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt 3240gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc 3300attgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca 3360gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata 3420gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg 3480ggcggagcct atggaaaaac gccagcaacg cggccttttt acggtcctgg ccttttgctg 3540gccttttgct cacatgtctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc 3600gcctttgagt gagctgatac cgctcgccgc agccgaaccg accgagcgca gcgagtcagt 3660gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat 3720tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc 3780aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc 3840tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacat 3900gattaccg 3908 25 41 DNA Artificial Sequence Synthetic expression vectorfragment 25 gatccttatt agatctcacc atcaccatca ccattgttga g 41 26 41 DNAArtificial Sequence Synthetic expression vector fragment 26 tcgactcaacaatggtgatg gtgatggtga gatctaataa g 41 27 3878 DNA Artificial SequenceSynthetic expression vector 27 ttgcaggaca ggatgtggtg cccgatgtgactagctcttt gctgcaggcc gtcctatcct 60 ctggttccga taagagaccc agaactccggccccccaccg cccaccgcca cccccataca 120 tatgtggtac gcaagtaaga gtgcctgcgcatgccccatg tgccccacca agagctttgc 180 atcccataca agtccccaaa gtggagaaccgaaccaattc ttcgcgggca gaacaaaagc 240 ttctgcacac gtctccactc gaatttggagccggccggcg tgtgcaaaag aggtgaatcg 300 aacgaaagac ccgtgtgtaa agccgcgtttccaaaatgta taaaaccgag agcatctggc 360 caatgtgcat cagttgtggt cagcagcaaaatcaagtgaa tcatctcagt gcaactaaag 420 gggaattcga gctcggtacc cggggatccttattagatct caccatcacc atcaccattg 480 ttgagtcgac ctgcaggcat gcaagctattcgatgcacac tcacattctt ctcctaatac 540 gataataaaa ctttccatga aaaatatggaaaaatatatg aaaattgaga aatccaaaaa 600 actgataaac gctctactta attaaaatagataaatggga gcggcaggaa tggcggagca 660 tggccaagtt cctccgccaa tcagtcgtaaaacagaagtc gtggaaagcg gatagaaaga 720 atgttcgatt tgacgggcaa gcatgtctgctatgtggcgg attgcggagg aattgcactg 780 gagaccagca aggttctcat gaccaagaatatagcggtga gtgagcggga agctcggttt 840 ctgtccagat cgaactcaaa actagtccagccagtcgctg tcgaaactaa ttaagttaat 900 gagtttttca tgttagtttc gcgctgagcaacaattaagt ttatgtttca gttcggctta 960 gatttcgctg aaggacttgc cactttcaatcaatacttta gaacaaaatc aaaactcatt 1020 ctaatagctt ggtgttcatc tttttttttaatgataagca ttttgtcgtt tatacttttt 1080 atatttcgat attaaaccac ctatgaagtctattttaatc gccagataag caatatattg 1140 tgtaaatatt tgtattcttt atcaggaaattcagggagac gggaagttac tatctactaa 1200 aagccaaaca atttcttaca gttttactctctctactcta gagtagcttg gcactggccg 1260 tcgttttaca acgtcgtgac tgggaaaaccctggcgttac ccaacttaat cgccttgcag 1320 cacatccccc tttcgccagc tggcgtaatagcgaagaggc ccgcaccgat cgcccttccc 1380 aacagttgcg cagcctgaat ggcgaatggcgcctgatgcg gtattttctc cttacgcatc 1440 tgtgcggtat ttcacaccgc atatggtgcactctcagtac aatctgctct gatgccgcat 1500 agttaagcca gccccgacac ccgccaacacccgctgacgc gccctgacgg gcttgtctgc 1560 tcccggcatc cgcttacaga caagctgtgaccgtctccgg gagctgcatg tgtcagaggt 1620 tttcaccgtc atcaccgaaa cgcgcgagacgaaagggcct cgtgatacgc ctatttttat 1680 aggttaatgt catgataata atggtttcttagacgtcagg tggcactttt cggggaaatg 1740 tgcgcggaac ccctatttgt ttatttttctaaatacattc aaatatgtat ccgctcatga 1800 gacaataacc ctgataaatg cttcaataatattgaaaaag gaagagtatg agtattcaac 1860 atttccgtgt cgcccttatt cccttttttgcggcattttg ccttcctgtt tttgctcacc 1920 cagaaacgct ggtgaaagta aaagatgctgaagatcagtt gggtgcacga gtgggttaca 1980 tcgaactgga tctcaacagc ggtaagatccttgagagttt tcgccccgaa gaacgttttc 2040 caatgatgag cacttttaaa gttctgctatgtggcgcggt attatcccgt attgacgccg 2100 ggcaagagca actcggtcgc cgcatacactattctcagaa tgacttggtt gagtactcac 2160 cagtcacaga aaagcatctt acggatggcatgacagtaag agaattatgc agtgctgcca 2220 taaccatgag tgataacact gcggccaacttacttctgac aacgatcgga ggaccgaagg 2280 agctaaccgc ttttttgcac aacatgggggatcatgtaac tcgccttgat cgttgggaac 2340 cggagctgaa tgaagccata ccaaacgacgagcgtgacac cacgatgcct gtagcaatgg 2400 caacaacgtt gcgcaaacta ttaactggcgaactacttac tctagcttcc cggcaacaat 2460 taatagactg gatggaggcg gataaagttgcaggaccact tctgcgctcg gcccttccgg 2520 ctggctggtt tattgctgat aaatctggagccggtgagcg tgggtctcgc ggtatcattg 2580 cagcactggg gccagatggt aagccctcccgtatcgtagt tatctacacg acggggagtc 2640 aggcaactat ggatgaacga aatagacagatcgctgagat aggtgcctca ctgattaagc 2700 attggtaact gtcagaccaa gtttactcatatatacttta gattgattta aaacttcatt 2760 tttaatttaa aaggatctag gtgaagatcctttttgataa tctcatgacc aaaatccctt 2820 aacgtgagtt ttcgttccac tgagcgtcagaccccgtaga aaagatcaaa ggatcttctt 2880 gagatccttt ttttctgcgc gtaatctgctgcttgcaaac aaaaaaacca ccgctaccag 2940 cggtggtttg tttgccggat caagagctaccaactctttt tccgaaggta actggcttca 3000 gcagagcgca gataccaaat actgtccttctagtgtagcc gtagttaggc caccacttca 3060 agaactctgt agcaccgcct acatacctcgctctgctaat cctgttacca gtggctgctg 3120 ccagtggcga taagtcgtgt cttaccgggttggactcaag acgatagtta ccggataagg 3180 cgcagcggtc gggctgaacg gggggttcgtgcacacagcc cagcttggag cgaacgacct 3240 acaccgaact gagataccta cagcgtgagcattgagaaag cgccacgctt cccgaaggga 3300 gaaaggcgga caggtatccg gtaagcggcagggtcggaac aggagagcgc acgagggagc 3360 ttccaggggg aaacgcctgg tatctttatagtcctgtcgg gtttcgccac ctctgacttg 3420 agcgtcgatt tttgtgatgc tcgtcaggggggcggagcct atggaaaaac gccagcaacg 3480 cggccttttt acggtcctgg ccttttgctggccttttgct cacatgtctt tcctgcgtta 3540 tcccctgatt ctgtggataa ccgtattaccgcctttgagt gagctgatac cgctcgccgc 3600 agccgaaccg accgagcgca gcgagtcagtgagcgaggaa gcggaagagc gcccaatacg 3660 caaaccgcct ctccccgcgc gttggccgattcattaatgc agctggcacg acaggtttcc 3720 cgactggaaa gcgggcagtg agcgcaacgcaattaatgtg agttagctca ctcattaggc 3780 accccaggct ttacacttta tgcttccggctcgtatgttg tgtggaattg tgagcggata 3840 acaatttcac acaggaaaca gctatgacatgattaccg 3878 28 47 DNA Artificial Sequence Synthetic expression vectorfragment 28 gatccttatt agatctgctt ggcgccatcc tcaatttggg ggttgag 47 29 47DNA Artificial Sequence Synthetic expression vector fragment 29tcgactcaac ccccaaattg aggatggcgc caagcagatc taataag 47 30 3883 DNAArtificial Sequence Synthetic expression vector 30 ttgcaggaca ggatgtggtgcccgatgtga ctagctcttt gctgcaggcc gtcctatcct 60 tggttccgat aagagacccagaactccggc cccccaccgc ccaccgccac ccccatacat 120 atgtggtacg caagtaagagtgcctgcgca tgccccatgt gccccaccaa gagctttgca 180 tcccatacaa gtccccaaagtggagaaccg aaccaattct tcgcgggcag aacaaaagct 240 tctgcacacg tctccactcgaatttggagc cggccggcgt gtgcaaaaga ggtgaatcga 300 acgaaagacc cgtgtgtaaagccgcgtttc caaaatgtat aaaaccgaga gcatctggcc 360 aatgtgcatc agttgtggtcagcagcaaaa tcaagtgaat catctcagtg caactaaagg 420 ggaattcgag ctcggtacccggggatcctt attagatctg cttggcgcca tcctcaattt 480 gggggttgag tcgacctgcaggcatgcaag ctattcgatg cacactcaca ttcttctcct 540 aatacgataa taaaactttccatgaaaaat atggaaaaat atatgaaaat tgagaaatcc 600 aaaaaactga taaacgctctacttaattaa aatagataaa tgggagcggc aggaatggcg 660 gagcatggcc aagttcctccgccaatcagt cgtaaaacag aagtcgtgga aagcggatag 720 aaagaatgtt cgatttgacgggcaagcatg tctgctatgt ggcggattgc ggaggaattg 780 cactggagac cagcaaggttctcatgacca agaatatagc ggtgagtgag cgggaagctc 840 ggtttctgtc cagatcgaactcaaaactag tccagccagt cgctgtcgaa actaattaag 900 ttaatgagtt tttcatgttagtttcgcgct gagcaacaat taagtttatg tttcagttcg 960 gcttagattt cgctgaaggacttgccactt tcaatcaata ctttagaaca aaatcaaaac 1020 tcattctaat agcttggtgttcatcttttt ttttaatgat aagcattttg tcgtttatac 1080 tttttatatt tcgatattaaaccacctatg aagtctattt taatcgccag ataagcaata 1140 tattgtgtaa atatttgtattctttatcag gaaattcagg gagacgggaa gttactatct 1200 actaaaagcc aaacaatttcttacagtttt actctctcta ctctagagta gcttggcact 1260 ggccgtcgtt ttacaacgtcgtgactggga aaaccctggc gttacccaac ttaatcgcct 1320 tgcagcacat ccccctttcgccagctggcg taatagcgaa gaggcccgca ccgatcgccc 1380 ttcccaacag ttgcgcagcctgaatggcga atggcgcctg atgcggtatt ttctccttac 1440 gcatctgtgc ggtatttcacaccgcatatg gtgcactctc agtacaatct gctctgatgc 1500 cgcatagtta agccagccccgacacccgcc aacacccgct gacgcgccct gacgggcttg 1560 tctgctcccg gcatccgcttacagacaagc tgtgaccgtc tccgggagct gcatgtgtca 1620 gaggttttca ccgtcatcaccgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt 1680 tttataggtt aatgtcatgataataatggt ttcttagacg tcaggtggca cttttcgggg 1740 aaatgtgcgc ggaacccctatttgtttatt tttctaaata cattcaaata tgtatccgct 1800 catgagacaa taaccctgataaatgcttca ataatattga aaaaggaaga gtatgagtat 1860 tcaacatttc cgtgtcgcccttattccctt ttttgcggca ttttgccttc ctgtttttgc 1920 tcacccagaa acgctggtgaaagtaaaaga tgctgaagat cagttgggtg cacgagtggg 1980 ttacatcgaa ctggatctcaacagcggtaa gatccttgag agttttcgcc ccgaagaacg 2040 ttttccaatg atgagcacttttaaagttct gctatgtggc gcggtattat cccgtattga 2100 cgccgggcaa gagcaactcggtcgccgcat acactattct cagaatgact tggttgagta 2160 ctcaccagtc acagaaaagcatcttacgga tggcatgaca gtaagagaat tatgcagtgc 2220 tgccataacc atgagtgataacactgcggc caacttactt ctgacaacga tcggaggacc 2280 gaaggagcta accgcttttttgcacaacat gggggatcat gtaactcgcc ttgatcgttg 2340 ggaaccggag ctgaatgaagccataccaaa cgacgagcgt gacaccacga tgcctgtagc 2400 aatggcaaca acgttgcgcaaactattaac tggcgaacta cttactctag cttcccggca 2460 acaattaata gactggatggaggcggataa agttgcagga ccacttctgc gctcggccct 2520 tccggctggc tggtttattgctgataaatc tggagccggt gagcgtgggt ctcgcggtat 2580 cattgcagca ctggggccagatggtaagcc ctcccgtatc gtagttatct acacgacggg 2640 gagtcaggca actatggatgaacgaaatag acagatcgct gagataggtg cctcactgat 2700 taagcattgg taactgtcagaccaagttta ctcatatata ctttagattg atttaaaact 2760 tcatttttaa tttaaaaggatctaggtgaa gatccttttt gataatctca tgaccaaaat 2820 cccttaacgt gagttttcgttccactgagc gtcagacccc gtagaaaaga tcaaaggatc 2880 ttcttgagat cctttttttctgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct 2940 accagcggtg gtttgtttgccggatcaaga gctaccaact ctttttccga aggtaactgg 3000 cttcagcaga gcgcagataccaaatactgt ccttctagtg tagccgtagt taggccacca 3060 cttcaagaac tctgtagcaccgcctacata cctcgctctg ctaatcctgt taccagtggc 3120 tgctgccagt ggcgataagtcgtgtcttac cgggttggac tcaagacgat agttaccgga 3180 taaggcgcag cggtcgggctgaacgggggg ttcgtgcaca cagcccagct tggagcgaac 3240 gacctacacc gaactgagatacctacagcg tgagcattga gaaagcgcca cgcttcccga 3300 agggagaaag gcggacaggtatccggtaag cggcagggtc ggaacaggag agcgcacgag 3360 ggagcttcca gggggaaacgcctggtatct ttatagtcct gtcgggtttc gccacctctg 3420 acttgagcgt cgatttttgtgatgctcgtc aggggggcgg agcctatgga aaaacgccag 3480 caacgcggcc tttttacggtcctggccttt tgctggcctt ttgctcacat gtctttcctg 3540 cgttatcccc tgattctgtggataaccgta ttaccgcctt tgagtgagct gataccgctc 3600 gccgcagccg aaccgaccgagcgcagcgag tcagtgagcg aggaagcgga agagcgccca 3660 atacgcaaac cgcctctccccgcgcgttgg ccgattcatt aatgcagctg gcacgacagg 3720 tttcccgact ggaaagcgggcagtgagcgc aacgcaatta atgtgagtta gctcactcat 3780 taggcacccc aggctttacactttatgctt ccggctcgta tgttgtgtgg aattgtgagc 3840 ggataacaat ttcacacaggaaacagctat gacatgatta ccg 3883 31 879 DNA Homo Sapiens 31 gaattcatgggccacacacg gaggcaggga acatcaccat ccaagtgtcc atacctcaat 60 ttctttcagctcttggtgct ggctggtctt tctcacttct gttcaggtgt tatccacgtg 120 accaaggaagtgaaagaagt ggcaacgctg tcctgtggtc acaatgtttc tgttgaagag 180 ctggcacaaactcgcatcta ctggcaaaag gagaagaaaa tggtgctgac tatgatgtct 240 ggggacatgaatatatggcc cgagtacaag aaccggacca tctttgatat cactaataac 300 ctctccattgtgatcctggc tctgcgccca tctgacgagg gcacatacga gtgtgttgtt 360 ctgaagtatgaaaaagacgc tttcaagcgg gaacacctgg ctgaagtgac gttatcagtc 420 aaagctgacttccctacacc tagtatatct gactttgaaa ttccaacttc taatattaga 480 aggataatttgctcaacctc tggaggtttt ccagagcctc acctctcctg gttggaaaat 540 ggagaagaattaaatgccat caacacaaca gtttcccaag atcctgaaac tgagctctat 600 gctgttagcagcaaactgga tttcaatatg acaaccaacc acagcttcat gtgtctcatc 660 aagtatggacatttaagagt gaatcagacc ttcaactgga atacaaccaa gcaagagcat 720 tttcctgataacctgctccc atcctgggcc attaccttaa tctcagtaaa tggaattttt 780 gtgatatgctgcctgaccta ctgctttgcc ccaagatgca gagagagaag gaggaatgag 840 agattgagaagggaaagtgt acgccctgta taaggattc 879 32 738 DNA Homo Sapiens 32gaattcatgg gccacacacg gaggcaggga acatcaccat ccaagtgtcc atacctcaat 60ttctttcagc tcttggtgct ggctggtctt tctcacttct gttcaggtgt tatccacgtg 120accaaggaag tgaaagaagt ggcaacgctg tcctgtggtc acaatgtttc tgttgaagag 180ctggcacaaa ctcgcatcta ctggcaaaag gagaagaaaa tggtgctgac tatgatgtct 240ggggacatga atatatggcc cgagtacaag aaccggacca tctttgatat cactaataac 300ctctccattg tgatcctggc tctgcgccca tctgacgagg gcacatacga gtgtgttgtt 360ctgaagtatg aaaaagacgc tttcaagcgg gaacacctgg ctgaagtgac gttatcagtc 420aaagctgact tccctacacc tagtatatct gactttgaaa ttccaacttc taatattaga 480aggataattt gctcaacctc tggaggtttt ccagagcctc acctctcctg gttggaaaat 540ggagaagaat taaatgccat caacacaaca gtttcccaag atcctgaaac tgagctctat 600gctgttagca gcaaactgga tttcaatatg acaaccaacc acagcttcat gtgtctcatc 660aagtatggac atttaagagt gaatcagacc ttcaactgga atacaaccaa gcaagagcat 720tttcctgata acggattc 738 33 1002 DNA Homo Sapiens 33 gagctcatggatccccagtg cactatggga ctgagtaaca ttctctttgt gatggccttc 60 ctgctctctggtgctgctcc tctgaagatt caagcttatt tcaatgagac tgcagacctg 120 ccatgccaatttgcaaactc tcaaaaccaa agcctgagtg agctagtagt attttggcag 180 gaccaggaaaacttggttct gaatgaggta tacttaggca aagagaaatt tgacagtgtt 240 cattccaagtatatgggccg cacaagtttt gattcggaca gttggaccct gagacttcac 300 aatcttcagatcaaggacaa gggcttgtat caatgtatca tccatcacaa aaagcccaca 360 ggaatgattcgcatccacca gatgaattct gaactgtcag tgcttgctaa cttcagtcaa 420 cctgaaatagtaccaatttc taatataaca gaaaatgtgt acataaattt gacctgctca 480 tctatacacggttacccaga acctaagaag atgagtgttt tgctaagaac caagaattca 540 actatcgagtatgatggtat tatgcagaaa tctcaagata atgtcacaga actgtacgac 600 gtttccatcagcttgtctgt ttcattccct gatgttacga gcaatatgac catcttctgt 660 attctggaaactgacaagac gcggctttta tcttcacctt tctctataga gcttgaggac 720 cctcagcctcccccagacca cattccttgg attacagctg tacttccaac agttattata 780 tgtgtgatggttttctgtct aattctatgg aaatggaaga agaagaagcg gcctcgcaac 840 tcttataaatgtggaaccaa cacaatggag agggaagaga gtgaacagac caagaaaaga 900 gaaaaaatccatatacctga aagatctgat gaagcccagc gtgtttttaa aagttcgaag 960 acatcttcatgcgacaaaag tgatacatgt ttttaagggc cc 1002 34 751 DNA Homo Sapiens 34gagctcatgg atccccagtg cactatggga ctgagtaaca ttctctttgt gatggccttc 60ctgctctctg gtgctgctcc tctgaagatt caagcttatt tcaatgagac tgcagacctg 120ccatgccaat ttgcaaactc tcaaaaccaa agcctgagtg agctagtagt attttggcag 180gaccaggaaa acttggttct gaatgaggta tacttaggca aagagaaatt tgacagtgtt 240cattccaagt atatgggccg cacaagtttt gattcggaca gttggaccct gagacttcac 300aatcttcaga tcaaggacaa gggcttgtat caatgtatca tccatcacaa aaagcccaca 360ggaatgattc gcatccacca gatgaattct gaactgtcag tgcttgctaa cttcagtcaa 420cctgaaatag taccaatttc taatataaca gaaaatgtgt acataaattt gacctgctca 480tctatacacg gttacccaga acctaagaag atgagtgttt tgctaagaac caagaattca 540actatcgagt atgatggtat tatgcagaaa tctcaagata atgtcacaga actgtacgac 600gtttccatca gcttgtctgt ttcattccct gatgttacga gcaatatgac catcttctgt 660attctggaaa ctgacaagac gcggctttta tcttcacctt tctctataga gcttgaggac 720cctcagcctc ccccagacca cattggggcc c 751 35 1611 DNA Homo Sapiens 35gaattcatgg ctcccagcag cccccggccc gcgctgcccg cactcctggt cctgctcggg 60gctctgttcc caggacctgg caatgcccag acatctgtgt ccccctcaaa agtcatcctg 120ccccggggag gctccgtgct ggtgacatgc agcacctcct gtgaccagcc caagttgttg 180ggcatagaga ccccgttgcc taaaaaggag ttgctcctgc ctgggaacaa ccggaaggtg 240tatgaactga gcaatgtgca agaagatagc caaccaatgt gctattcaaa ctgccctgat 300gggcagtcaa cagctaaaac cttcctcacc gtgtactgga ctccagaacg ggtggaactg 360gcacccctcc cctcttggca gccagtgggc aagaacctta ccctacgctg ccaggtggag 420ggtggggcac cccgggccaa cctcaccgtg gtgctgctcc gtggggagaa ggagctgaaa 480cgggagccag ctgtggggga gcccgctgag gtcacgacca cggtgctggt gaggagagat 540caccatggag ccaatttctc gtgccgcact gaactggacc tgcggcccca agggctggag 600ctgtttgaga acacctcggc cccctaccag ctccagacct ttgtcctgcc agcgactccc 660ccacaacttg tcagcccccg ggtcctagag gtggacacgc aggggaccgt ggtctgttcc 720ctggacgggc tgttcccagt ctcggaggcc caggtccacc tggcactggg ggaccagagg 780ttgaacccca cagtcaccta tggcaacgac tccttctcgg ccaaggcctc agtcagtgtg 840accgcagagg acgagggcac ccagcggctg acgtgtgcag taatactggg gaaccagagc 900caggagacac tgcagacagt gaccatctac agctttccgg cgcccaacgt gattctgacg 960aagccagagg tctcagaagg gaccgaggtg acagtgaagt gtgaggccca ccctagagcc 1020aaggtgacgc tgaatggggt tccagcccag ccactgggcc cgagggccca gctcctgctg 1080aaggccaccc cagaggacaa cgggcgcagc ttctcctgct ctgcaaccct ggaggtggcc 1140ggccagctta tacacaagaa ccagacccgg gagcttcgtg tcctgtatgg cccccgactg 1200gacgagaggg attgtccggg aaactggacg tggccagaaa attcccagca gactccaatg 1260tgccaggctt gggggaaccc attgcccgag ctcaagtgtc taaaggatgg cactttccca 1320ctgcccatcg gggaatcagt gactgtcact cgagatcttg agggcaccta cctctgtcgg 1380gccaggagca ctcaagggga ggtcacccgc gaggtgaccg tgaatgtgct ctccccccgg 1440tatgagattg tcatcatcac tgtggtagca gccgcagtca taatgggcac tgcaggcctc 1500agcacgtacc tctataaccg ccagcggaag atcaagaaat acagactaca acaggcccaa 1560aaagggaccc ccatgaaacc gaacacacaa gccacgcctc cctgaggatc c 1611 36 1452DNA Homo Sapiens 36 gaattcatgg ctcccagcag cccccggccc gcgctgcccgcactcctggt cctgctcggg 60 gctctgttcc caggacctgg caatgcccag acatctgtgtccccctcaaa agtcatcctg 120 ccccggggag gctccgtgct ggtgacatgc agcacctcctgtgaccagcc caagttgttg 180 ggcatagaga ccccgttgcc taaaaaggag ttgctcctgcctgggaacaa ccggaaggtg 240 tatgaactga gcaatgtgca agaagatagc caaccaatgtgctattcaaa ctgccctgat 300 gggcagtcaa cagctaaaac cttcctcacc gtgtactggactccagaacg ggtggaactg 360 gcacccctcc cctcttggca gccagtgggc aagaaccttaccctacgctg ccaggtggag 420 ggtggggcac cccgggccaa cctcaccgtg gtgctgctccgtggggagaa ggagctgaaa 480 cgggagccag ctgtggggga gcccgctgag gtcacgaccacggtgctggt gaggagagat 540 caccatggag ccaatttctc gtgccgcact gaactggacctgcggcccca agggctggag 600 ctgtttgaga acacctcggc cccctaccag ctccagacctttgtcctgcc agcgactccc 660 ccacaacttg tcagcccccg ggtcctagag gtggacacgcaggggaccgt ggtctgttcc 720 ctggacgggc tgttcccagt ctcggaggcc caggtccacctggcactggg ggaccagagg 780 ttgaacccca cagtcaccta tggcaacgac tccttctcggccaaggcctc agtcagtgtg 840 accgcagagg acgagggcac ccagcggctg acgtgtgcagtaatactggg gaaccagagc 900 caggagacac tgcagacagt gaccatctac agctttccggcgcccaacgt gattctgacg 960 aagccagagg tctcagaagg gaccgaggtg acagtgaagtgtgaggccca ccctagagcc 1020 aaggtgacgc tgaatggggt tccagcccag ccactgggcccgagggccca gctcctgctg 1080 aaggccaccc cagaggacaa cgggcgcagc ttctcctgctctgcaaccct ggaggtggcc 1140 ggccagctta tacacaagaa ccagacccgg gagcttcgtgtcctgtatgg cccccgactg 1200 gacgagaggg attgtccggg aaactggacg tggccagaaaattcccagca gactccaatg 1260 tgccaggctt gggggaaccc attgcccgag ctcaagtgtctaaaggatgg cactttccca 1320 ctgcccatcg gggaatcagt gactgtcact cgagatcttgagggcaccta cctctgtcgg 1380 gccaggagca ctcaagggga ggtcacccgc gaggtgaccgtgaatgtgct ctccccccgg 1440 tatgagggat cc 1452 37 726 DNA Homo Sapiens 37gagctcatgg ttgctgggag cgacgcgggg cgggccctgg gggtcctcag cgtggtctgc 60ctgctgcact gctttggttt catcagctgt ttttcccaac aaatatatgg tgttgtgtat 120gggaatgtaa ctttccatgt accaagcaat gtgcctttaa aagaggtcct atggaaaaaa 180caaaaggata aagttgcaga actggaaaat tctgaattca gagctttctc atcttttaaa 240aatagggttt atttagacac tgtgtcaggt agcctcacta tctacaactt aacatcatca 300gatgaagatg agtatgaaat ggaatcgcca aatattactg ataccatgaa gttctttctt 360tatgtgcttg agtctcttcc atctcccaca ctaacttgtg cattgactaa tggaagcatt 420gaagtccaat gcatgatacc agagcattac aacagccatc gaggacttat aatgtactca 480tgggattgtc ctatggagca atgtaaacgt aactcaacca gtatatattt taagatggaa 540aatgatcttc cacaaaaaat acagtgtact cttagcaatc cattatttaa tacaacatca 600tcaatcattt tgacaacctg tatcccaagc agcggtcatt caagacacag atatgcactt 660atacccatac cattagcagt aattacaaca tgtattgtgc tgtatatgaa tgttctttaa 720ggatcc 726 38 657 DNA Homo Sapiens 38 gagctcatgg ttgctgggag cgacgcggggcgggccctgg gggtcctcag cgtggtctgc 60 ctgctgcact gctttggttt catcagctgtttttcccaac aaatatatgg tgttgtgtat 120 gggaatgtaa ctttccatgt accaagcaatgtgcctttaa aagaggtcct atggaaaaaa 180 caaaaggata aagttgcaga actggaaaattctgaattca gagctttctc atcttttaaa 240 aatagggttt atttagacac tgtgtcaggtagcctcacta tctacaactt aacatcatca 300 gatgaagatg agtatgaaat ggaatcgccaaatattactg ataccatgaa gttctttctt 360 tatgtgcttg agtctcttcc atctcccacactaacttgtg cattgactaa tggaagcatt 420 gaagtccaat gcatgatacc agagcattacaacagccatc gaggacttat aatgtactca 480 tgggattgtc ctatggagca atgtaaacgtaactcaacca gtatatattt taagatggaa 540 aatgatcttc cacaaaaaat acagtgtactcttagcaatc cattatttaa tacaacatca 600 tcaatcattt tgacaacctg tatcccaagcagcggtcatt caagacacag aggatcc 657 39 23 PRT Artificial SequenceSynthetic ovalbumin antigenic peptide 39 Gln Leu Glu Ser Ile Ile Asn PheGlu Lys Leu Thr Glu Trp Thr Ser 1 5 10 15 Ser Asn Val Met Glu Glu Arg 2040 10 PRT Artificial Sequence Synthetic vesicular stomatitis antigenicpeptide 40 Asp Leu Arg Gly Tyr Val Tyr Gln Gly Leu 1 5 10 41 9 PRTArtificial Sequence Synthetic HIV antigenic peptide 41 Phe Arg Ile GlyCys Arg His Ser Arg 1 5 42 9 PRT Artificial Sequence Synthetic HIVantigenic peptide 42 Ile Leu Lys Glu Pro Val His Gly Val 1 5 43 32 DNAArtificial Sequence Synthetic PCR primer (SPP) 43 atatggatcc tcaccatctcagggtgaggg gc 32 44 10 PRT Artificial Sequence Synthetic vesicularstomatitis antigenic peptide 44 Arg Gly Tyr Val Tyr Gln Gly Leu Lys Ser1 5 10 45 9 PRT Artificial Sequence Mus Musculus 45 Phe Ala Pro Gly AsnTyr Pro Ala Leu 1 5 46 8 PRT Artificial Sequence Mus musculus 46 Leu SerPro Phe Pro Phe Asp Leu 1 5 47 9 PRT Artificial Sequence Mus musculus 47Gln Leu Ser Pro Phe Pro Phe Asp Leu 1 5 48 9 PRT Artificial SequenceSynthetic Antigen 48 Ile Leu Lys Glu Pro Val His Gly Val 1 5 49 9 PRTArtificial Sequence Synthetic antigen 49 Tyr Met Asn Gly Thr Met Ser GlnVal 1 5 50 9 PRT Artificial Sequence Synthetic antigenic 50 Gly Ile LeuGly Phe Val Phe Thr Leu 1 5 51 10 PRT Artificial Sequence Syntheticantigenic 51 Phe Leu Pro Ser Asp Phe Phe Pro Ser Val 1 5 10 52 34 DNAArtificial Sequence PCR primer 52 tttagaattc accatggctt caacccgtgc caag34 53 31 DNA Artificial Sequence PCR primer 53 tttagtcgac tcagggaggtggggcttgtc c 31 54 34 DNA Artificial Sequence PCR primer 54 tttagaattcaccatggctt gcaattgtca gttg 34 55 31 DNA Artificial Sequence PCR primer55 tttagtcgac ctaaaggaag acggtctgtt c 31 56 36 DNA Artificial SequencePCR primer 56 tttagaattc accatggacc ccagatgcac catggg 36 57 34 DNAArtificial Sequence PCR primer 57 tttagtcgac tcactctgca tttggttttg ctga34 58 33 DNA Artificial Sequence PCR Primer 58 acccttgaat ccatgggccacacacggagg cag 33 59 39 DNA Artificial Sequence PCR Primer 59 attaccggatccttatacag ggcgtacact ttcccttct 39 60 33 DNA Artificial Sequence PCRprimer 60 acccttgagc tcatggatcc ccagtgcact atg 33 61 42 DNA ArtificialSequence PCR primer 61 attacccccg ggttaaaaac atgtatcact tttgtcgcat ga 4262 36 DNA Sequence Listing PCR primer 62 acccttgagc tcatggttgctgggagcgac gcgggg 36 63 42 DNA Sequence Listing PCR primer 63 attaccggatccttaaagaa cattcatata cagcacaata ca 42 64 36 DNA Squence Listing PCRprimer 64 acccttgaat tcatggctcc cagcagcccc cggccc 36 65 39 DNAArtificial Sequence PCR primer 65 attaccggat cctcagggag gcgtggcttgtgtgttcgg 39

We claim:
 1. A method for activating CD8⁺ T-cells against a selectedpeptide, the method comprising: a) providing a synthetic antigenpresenting matrix comprising (i) a support comprising a fragment of aDrosophila cell; (ii) extracellular portion of at least one MHC class Imolecule capable of binding to a selected peptide and being linked tothe support; and (iii) at least one assisting molecule linked to thesupport such that the extracellular portions of the MHC and assistingmolecules are present in sufficient numbers to activate a population ofT lymphocytes specific for said peptide when the peptide is bound to thepeptide binding extracellular portion of the MHC molecule; wherein saidassisting molecule is selected from the group consisting of B7.1, B7.2,ICAM-1, ICAM-2, ICAM-3, LFA-3 and anti-CD28 antibody; said MHC moleculebeing provided by said Drosophila cell and comprising a heavy chain anda β-2 microglobulin; and b) contacting the matrix with the CD8⁺ T-cells.2. A method for generating cytotoxic CD8⁺ T-cells against a selectedpeptide, the method comprising: a) providing a synthetic antigenpresenting matrix comprising (i) a support comprising a fragment of aDrosophila cell; (ii) extracellular portion of at least one MHC class Imolecule capable of binding to a selected peptide and being linked tothe support; and (iii) at least one assisting molecule linked to thesupport such that the extracellular portions of the MHC and assistingmolecules are present in sufficient numbers to activate a population ofT lymphocytes specific for said peptide when the peptide is bound to thepeptide binding extracellular portion of the MHC molecule; wherein saidassisting molecule is selected from the group consisting of B7.1, B7.2,ICAM-1, ICAM-2, ICAM-3, LFA-3 and anti-CD28 antibody; said MHC moleculebeing provided by said Drosophila cell and comprising a heavy chain anda β-2 microglobulin; and b) contacting naive CD8⁺ T-cells with thematrix in vitro.
 3. The method of claim 2 wherein the support is a cell.4. The method of claim 2 wherein the assisting molecule is costimulatorymolecule B7.1 or B7.2.
 5. The method of claim 2 wherein the assistingmolecule is an anti-CD28 antibody.
 6. The method of claim 2 wherein theassisting molecule is an adhesion molecule selected from the groupconsisting of ICAM-1, ICAM-2, ICAM-3, and LFA-3.
 7. The method of claim2 wherein the assisting molecule is costimulatory molecule B7.1.
 8. Themethod of claim 2 wherein the assisting molecule is costimulatorymolecule B7.2.
 9. The method of claim 2 wherein the assisting moleculesare ICAM-1 and B7.1.
 10. The method of claim 2 wherein the assistingmolecules are ICAM-1 and B7.2.